Review
Electrochemical Biosensors for Cancer Biomarker Detection Jianping Li,*+a, b Shuhuai Li,+b Catherine F. Yang*+a a
Department of Chemistry and Biochemistry, Rowan University, Glassboro, NJ 08028, USA tel.: + (856)256-5455 b College of Chemistry and Bioengineering, Guilin University of Technology, Guilin, 541004, P. R. China *e-mail:
[email protected];
[email protected] Received: August 17, 2012;& Accepted: September 12, 2012 Abstract Cancer is still one of the leading causes of death in the world. There are over 200 types of cancers currently known according to the National Cancer Institute. However, early diagnosis continues to be an important integral part of cancer treatment even though many advances in therapeutics have been made in the past decade. Quick diagnosis and early prevention are critical for the control of the disease status. Biomarkers are commonly indicative of a particular disease process and the cancer biomarkers are also widely used in oncology to help detecting the presence of various carcinomas. The detection of cancer biomarkers plays an important role in clinical diagnoses and evaluation of treatment for patients. Many immunoassay methods are developed for detection of cancer biomarkers. As the detection devices are normally viewed with high sensitivity, simple preparation and rapid response, electrochemical biosensors are increasingly used for the detection of cancer markers. This review describes the status, the latest research and trends of electrochemical sensors in the quantitation of cancer markers in recent years. In particular, the strategy to improve the sensitivities of the electrochemical biosensors by the aid of enzymatic amplification, nanoparticle amplification, ultilization of magnetic microspheres etc. is described herein. At last, we discuss some special features and limitations associated with the described systems that summarize the application and the development prospects of electrochemical immunoassay technology. Keywords: Biomarkers, Electrochemical biosensors, Cancer
DOI: 10.1002/elan.201200447
1 Biomarkers in Early Diagnosis of Cancer Diagnosis is the process for translation of data gathered by clinical examination into an organized, classified definition of the conditions presented. The early and quick diagnosis of cardiovascular disease is extremely important not only for patient survival, but also for saving cost and a great deal of time in successful prognosis of the diseases. To find clinical biomarkers is crucial for the early detection of cancers, design of individual therapies, and to identify underlying processes involved in the disease [1]. Biomarkers are the chemical substance related with the elevation of malignant tumors which are found in the blood, urine, or body tissues. Biomarkers normally are produced directly by the embryonic tissue or tumor tissue [2]. According to the National Institutes of Health, a biomarker is a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmaceutical responses to a therapeutic intervention [3]. Biomarkers indicate changes in the expression of a protein that is correlated to risk or progression of a disease or its response to treatment, and that can be measured in tissues or in the blood. As a result, biomarkers can be specific cells, molecules or genes, gene products, enzymes or hormones [4]. Biomarkers are important as molecular signposts of the Electroanalysis 2012, 24, No. 12, 2213 – 2229
physiological state of a cell at a specific time. So a biomarker is objectively measured and evaluated as an indicator of normal biologic or pathogenic processes to a therapeutic intervention. Each biomarker is indicative of a particular disease process and the cancer biomarkers are used in oncology to help detect the presence of carcinomas. Biomarkers can be measured in biological media such as tissues, cells, or fluids. To maximize the usefulness and minimize the cost for screening, it is advantageous for these biomarkers to be measurable. There are many different cancer biomarkers, which can be classified into the types of embryonic antigen, carbohydrate antigen, enzymatic tumor markers, isoenzyme tumor markers, protein cancer markers, oncogene-related cancer biomarkers and hormone related cancer markers [5]. It is possible to obtain information about the tumor on the tissue histogenesis, the cell differentiation, cell functionalization by the qualitative and quantitative analysis of cancer markers, which provide the basis of the early detection or diagnosis of primary cancer, cancer classification, prognosis, and therapeutic guidelines [6,7]. Therefore, the detection of cancer markers is of great clinical significance [8,9].
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2 Strategy of Designing Electrochemical Sensors for Biomarkers Detection 2.1 Introduction of Electroanalysis and Electrochemical Biosensors in Biomarkers Detection The classical methods (such as ELISA) for diagnosis of cancer may take several hours, or even days from when tests are ordered to when results are received. These methods can be tedious, time consuming and often require extra care and expensive instruments. This especially makes early diagnosis of cancer more difficult for the cancer patients who are admitted to an emergency department. Therefore, measurement of carcinomatous markers is critical in assisting the diagnosis of cancer. A more sensitive and rapid technology platform is urgently needed to fulfill the rapid diagnosis requirements in cancer marker detection during early stages of the disease [10]. At present, many research studies have been carried out in the detection of cancer markers. These mainly included spectrophotometric or optical methods [11], fluorescence immunoassay [12,13], chemiluminescence analysis [14,15], electrochemical analysis [16–18], radioimmunoassay [19], capillary electrophoresis and chromatographic analysis [20]. One of the key challenges in detecting biomarkers in cancer is lack of sensitivity. Electrochemical analysis is one of the most sensitive methods for detecting inorganic, organic and even biologic substances. It is also suitable when used in the assay of cancer biomarkers [21,22]. The application of electrochemical immunoassay is likely the most promising way to solve some of the problems concerning sensitivity, speed, selectivity and economic (one-step) measurements because an effective combination of immunochemistry coupled with electrochemistry could provide the basis of direct electrical detection for a wide range of analytes with specificity and great sensitivity [23]. In consideration of its portability, low cost and high sensitivity, electrochemical sensors have become an attractive alternative to help in rapid diagnosis, providing better intervention and reducing the test-time of dissemination, which is highly beneficial in reducing patient stress[24]. 2.2 The Immobilization of Sensitive Elements within the Film of Electrochemical Sensors Electrochemical analysis method widely used in bioassay and medical testing has high sensitivity, low detection limit, and a wide linear detection range. It is simple, convenient and utilizes minimal instrumentation [25]. Electrochemical immunosensors provide the combined characterizations of high sensitivity of electrochemical analysis. The specific recognition of immunochemical reaction is suitable for trace analysis in biochemical substrates [26]. The advantages of sensitive simple instrumentation, and simultaneous detection of electrochemical immunosensors were demonstrated when they were applied to 2214
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the detection of cancer markers [27,28]. As early as 1980, Aizawa et al. [29] fixed AFP antibody on the carboxylic acid of a cellulose membrane and adhered the membrane onto the permeable membrane surface of a Clark-type oxygen electrode to construct an AFP immunosensor. The second antibody of AFP labelled by HRP, AFP could be determined in the concentration of 10 11–10 8 g/ mL by competitive immunoreaction. Subsequently, electrochemical immunosensors were applied in the detection of various cancer biomarkers. To prepare electrochemical biosensors, the immobilization of the biomolecule and the strategy to improve the sensitivity should be a key factor in sensor design and construction. There are many types of signals of electrochemical sensors that can be recorded during the detection of cancer biomarkers [30,31]. According to the signals measured, the electrochemical immunoassay can be divided into several categories according to conductivity method [32], potentiometric method [33,34], amperometric method [35,36], electrochemical luminescence (ECL) method [37,38], AC impedance and capacitance method [39,40], gravimetric method [41], piezoelectric quartz crystal method [42], surface plasmon resonance [43], etc. Among these methods, the electrochemical luminescence method is relatively more sensitive than others. The fabrication of the sensitive membrane is critical to the development of electrochemical immunosensors. The methods to immobilize the immunological reagents (i.e. antibodies) in the sensitive membrane usually include the methods of physical adsorption, covalent binding, crosslinking, self-assembly, embedding and Langumir–Biodgett (L-B) membrane. The physical adsorption method is to adsorb biological components on the insoluble inert carrier by molecules with polar bond, hydrogen bond and hydrophobic bond. The method is simple and generates little effect on biological activity. However, the electrode is not stable enough and the sensitive membrane is easy to fall off, due to weak interaction between biological molecules and the carrier. Nanoparticles have attracted growing attention in immobilization of biological macromolecules such as immunoreagents and immune protein, due to the large surface area, strong adsorption capacity. Mao et al. [44] used the Nafion membrane modified titanium dioxide nanoparticles (TiO2), human chorionic gonadotrophin antibody was adsorbed on it by the electrostatic interaction, and then Ru(bpy)32 + was bonded on the surface of TiO2 nanoparticles by ion exchange to successfully prepare of Ru-Nafion@TiO2 antibody modified gold electrode. Human chorionic gonadotropin was determined with a detection limit of 0.007 mU/mL by sandwich reaction. Covalent bond-combined technique is that of an immobilizing antibody (or antigen) which is linked onto a transducer surface by covalent bond. The immobilized antigen or antibody by covalent bond is relatively stable, but the activity of the antigen/antibody might be reduced and the procedure of electrode modification is complex.
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Fig. 1. Representation of ECL-based SWCNT immunosensors after addition of PSA and the Ru(bpy)32 + -silica-Ab2 nanoparticles. (Adapted from Sardesai et al. [45])
Sardesai et al. [45] reported an electrochemiluminescent immunosensor combining single-wall carbon nanotube (SWCNT) for a sensitive detection of cancer biomarker prostate-specific antigen (PSA), although there is a controversy on the clinical relevance on the PSA level after chemotherapy. They designed a sandwich immunoassay for PSA by first chemically attaching capture antibodies (Ab1) on SWCNT forests on pyrolytic graphite disks (Figure 1). The immunosensor was then incubated with serum and PSA was captured on the sensor surface. After washing with nonspecific binding blockers, the Ru(bpy)32 + -silica-secondary antibody (Ab2) nanoparticle bioconjugate was added and then ECL was measured. The detection limit of the sensor was 40 pg/mL. Cross-linking polymerization between functional monomers (also between functional monomer and biomolecules) was carried out to form the polymer with mesh structures. The biomolecules such as antigen, antibody and enzyme were embedded in the polymer. The activity of biological materials can likely be maintained by chemical cross-linking method. Sarkar [46] developed a simple and cost effective technique for identification and monitoring of prostate cancer using amperometric immunosensor. The immunosensor was constructed by anti-PSA and GOD immobilized on hydroxyethyl cellulose and carbon powder containing a 5 % rhodium modified electrode. HRP labeled antibody was used as a tracer. The electrochemical response was observed due to enzymatic reaction via a sandwich immunoassay. The strategy involved signal amplification by regeneration of the enzymatic substrate within the membrane layer of the electrode and the accumulation of the redox mediators (I2/I ). Self-assembly method is commonly used in recent years to immobilize the electrode sensitive materials by electrostatic interaction with oppositely charged compounds. Miao et al. [47] established an anodic electrogenerated chemiluminescence with tri-n-propylamine (TPrA) as a co-reactant to determine C-reactive protein (CRP) using Ru(bpy)32 + labels. Biotinylated anti-CRP species Electroanalysis 2012, 24, No. 12, 2213 – 2229
were immobilized onto the Au substrate precovered with a layer of avidin linked covalently via the reaction between avidin and a mixed thiol monolayer of 3-mercaptopropanoic acid and 16-mercaptohexadecanoic acid on Au electrodes. CRP and anti-CRP tagged with Ru(bpy)32 + labels were then conjugated onto the surface layer. The ECL peak intensity was linearly proportional to the concentration of analyte CRP over the range 1–24 mg/mL. However, the sensitivity was restricted, due to the limitation of the electrode surface for antibodies immobilization. For the embedding method, the electrode sensitive materials were embedded in the three-dimensional structural network of polymer. The merit of this method is that the immobilized biological molecules are avoided to be effused out. However, polymer gel or semi-permeable membrane utilized in the method is not conductive to the diffusion of macromolecular substrate and their products and the free radicals generated in the process of polymer film formation may do harm to the biological components embedded. Liu et al. [48] prepared a hydrophilic and nontoxic colloidal silica nanoparticle/titania sol-gel composite membrane on a gold electrode via a chemical vapor deposition method for encapsulation of carcinoembryonic antibody (anti-CEA) to construction of an electrochemical immunosensor. The presence of silica nanoparticles provided a congenial microenvironment for adsorbed biomolecules. The formation of immunoconjugate by a simple one-step immunoreaction between CEA in sample solution and the immobilized anti-CEA introduced the change in the potential. The immunosensor exhibited wide linear range for CEA determination from 1.5 to 240 ng/mL with a detection limit of 0.5 ng/mL. This composite membrane could be used efficiently for the entrapment of biomarkers. LB membrane technology is the molecular level plateau for production of single-layer or multilayer molecular film which is used to emerge up the immune reagents. LB technique appears to be quite suitable for generating bio-specific surfaces and it has the potential application for fabricating biosensors. Hou et al. [49] deposited the LB films of immunoglobulin G and amphiphile onto hydrophobic silver surface previously modified by 1-octadecanethiol (ODT) SAMs. The immunosensor obtained exhibits response to immunoglobulin G in a linear dynamic range from 200 to 1000 ng/mL with a good specificity.
3 Strategy to Improve the Sensitivity of Electrochemical Biosensors Due to the extremely low concentration of cancer biomarkers in tissue or blood, the sensitivity of the sensors is an important indicator to evaluate the newly developed immunosensors for cancer diagnosis. Continuously improving the sensitivity of the sensor is the requirement of analysis testing and is currently the focus of this study.
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The methods for improvement of the sensitivity of immunosensors were adopted by the measurement signal amplification via enzymatic reaction, magnetic microspheres, application of nanomaterials, controlled release of electroactive regents and polymer in the film of the sensors.
3.1 Enzymatic Amplification Enzymes are protein biocatalysts that catalyze specific biological reactions; enzyme-catalyzed reaction can produce exponential amplification effects which are largely applied in electrochemical sensors detection. It is a common strategy for construction of chemical biosensors by the use of enzymatic amplification. During the preparation of immunosensors, oxidase i.e. HRP and GOD are often labeled on a second antibody. The enzymatic production or the subsequent productions are electrochemically measured. Due to the amplification cycle of enzymatic reaction, trace amount of the analyte of antigen would bring about enlargement of the concentration of the measured substrates [50]. When the loaded amount of enzyme increases, the sensitivity of the determination can be promoted significant. For example, Dhawan [51] designed a new multienzyme marked electrochemical biosensor. Spherical polystyrene microparticles expressing a large number of highly reactive functional groups were chemically engineered to generate antibody-enzyme conjugates as novel signal amplification systems. Chemically modified goat anti-human IgG and HRP were attached to streptavidin microparticles. The numbers of HRP molecules/microparticle were further increased by coupling HRP to primary amines on N-terminal biotinylated or bromoacetylated polypeptides containing 20 lysine residues prior to conjugation with microparticles. The antibody-poly-HRP immunoconjugates contained an estimated number of 105 HRP/streptavidin microparticle and 106 HRP/amino microparticle, respectively. These microparticle immunoconjugates efficiently bound to plasma anti-HIV antibodies that had been captured by HIV antigens on magnetic microparticles, produced a detection signal with 5–8 times more sensitivity as compared to conventional HRP-conjugated goat anti-human IgG.
cal, electrochemical properties, often used for signal amplification of the detection by immunosensors. Carbon nanotube (CNT) is an one-dimensional (1-D) nanosized fibrous and tubular material. CNTs, including single-wall carbon nanotubes (SWCNT) and multiwall carbon nanotubes (MWCNT), have generated a considerable interest owing to their outstanding structure-dependent electronic and mechanical properties, and special chemical and physical properties which are capable of developing superior electrochemical sensing devices [56,57]. Ruslings group has made great efforts to improving sensitivity of electrochemical sensors by utilization of nanophase materials [58–61]. For example, they employed SWNT forest with multilabel secondary antibody-nanotube bioconjugates to construct a platform for highly sensitive detection of a cancer biomarker in serum and tissue lysates [62]. Greatly amplified sensitivity was attained by using bioconjugates featuring HRP labels and secondary antibodies (Ab2) linked to CNT at a high HRP/Ab2 ratio (Figure 2). This approach provided a detection limit of 4 pg/mL (100 amol/mL). Gold nanoparticles (AuNPs) have attracted considerable scientific interest because of their broad application in preparing biosensors, such as carriers of enzyme and antibody and enhancement of the conductivity and the affinity to bioactive materials [63,64]. Ou et al. [65] developed an amperometric immunosensor for the detection of CEA based on layer-by-layer assembly of AuNPs, multiwalled carbon nanotubes-thionine (MWNTs-THI) and chitosan on 3-mercaptopropanesulfonic sodium (MPS)modified gold electrode surface by electrostatic adsorption. The immunosensor was highly sensitive to CEA with a detection limit of 0.01 ng/mL, due to the addition of gold nanoparticles and carbon nanotubes. Graphene represents a conceptually new class of nanomaterials with a single layer 2-D sheet of carbon atoms in a hexagonal configuration which is available for electron transport [66–68]. It has attracted significant interest in several applications including biosensors. Recently, an explosive growth in work reported is related to the use of
3.2 Amplification via Nanoparticles Application of nanoparticles in biosensors has received great attentions in recent years due to their many desirable properties [52–54]. Nanoparticles are the nanoscaled materials with the various sizes at the 1–100 nm scale. Nanoparticles used in fabrication of immunosensors including carbon nanotube, grapheme, metal nanoparticles and quantum dot. Mostly, signal amplification in electrochemical immunosensors depends on the type of electroactive labels used, which are often enzyme-functionalized nanoparticles [55]. The nanoparticle surface is easily to be modified and functionalized, with good optical, electri2216
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Fig. 2. Illustration of detection principles of the immunosensor after treating with HRP-SWCNT-Ab2 to obtain amplification by providing numerous enzyme labels per binding event. (Adapted from Yu et al. [61])
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Fig. 3. Schematic representation of (A) the preparation of tracing tag and labeled Ab2, and (B) Immunosensor fabrication and sandwich-type immunoassay procedure (Adapted from Lin et al. [69])
graphene-based electrodes for electrochemical sensing, due to its large surface area and good electrical conductivity. The sensitivities of these biosensors were promoted significantly. For example, Lin et al. [69] constructed a CEA immunosensor with triple signal amplification by using graphene to modify immunosensor surface for accelerating electron transfer, poly(styrene-co-acrylic acid) microbead carried AuNPs as a tracing tag to label signal antibody (Ab2) and AuNPs induced silver deposition for anodic stripping analysis. With a sandwich-type immunoreaction, the AuNPs labeled Ab2 was captured on the surface of the immunosensor to further induce a silver deposition process. The linear stripping voltammetric method was used to monitor the deposited silver nanoparticles derived from the immunoreaction (Figure 3). The triple signal amplification of graphene, nanoparticles of gold and silver greatly enhanced the sensitivity for biomarker detection, and the detection limit was down to 0.12 pg/mL. Anodic striping voltammetry (ASV) is known for its high sensitivity. Immunoassay combined metal nanoparticles and ASV has been observed to have extremely high sensitivity [70,71]. Sardesai et al. [72] developed a multiplexed electrochemical immunoassay method for simultaneous ultrasensitive measurement of tumor markers based on ASV analysis of silver nanoparticles. A disposable array was prepared by covalently immobilizing antibodies on chitosan modified screen-printed carbon electrodes. After a sandwich-type immunoreaction, AuNPs labeled antibodies were captured onto an immunosensor Electroanalysis 2012, 24, No. 12, 2213 – 2229
surface to induce the silver catalytic deposition from a silver solution. The deposited silver nanoparticles were measured by ASV. The analytical sensitivity of protein markers was enhanced by the catalytic deposition. Carcinoembryonic antigen and a-fetoprotein were selected as analytes. The multiplexed immunoassay showed the detection limits of 3.5 and 3.9 pg/mL respectively, which give a promising potential in multianalyte determination for clinical application. By combination of amplifications produced from enzymatic reaction and nanoparticles, Ju and his co-workers have also made a successive attempt [73–76]. They prepared a glucose oxidase-functionalized nanocomposite with coating layer-by-layer colloidal Prussian blue (PB), gold nanoparticles, and captured antibodies on screenprinted carbon electrodes to construct an ultrasensitive multiplexed sandwich-type immunosensor array [77]. The colloidal PB acted as a mediator to catalyze the reduction of H2O2 produced in the GOD enzymatic reaction. Both the high-content glucose oxidase and carbon nanotubes in the tracer amplified the detectable signal of the immunoassay. Related references on the biosensors based on nanoparticles and magnetic particles were shown in Table 1. 3.3 Magnetic Microspheres Magnetic beads, microspheres and nanoparticles have been widely used in electrochemical immunoassay because of their unique properties of superparamagnetism,
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Table 1. Some study related to the signal amplification based on nanoparticle. Target Composition of sensitive film
Detection method
Detection limit
Linear range
Ref.
PSA IL-6 PSA IL-6 AFP
CNT/Au-Ab1/Ag/Ab2-HRP SWNT-Ab1/Ag/Ab2-HRP SWCNT forests-Ab1/Ag/Ab2-Ru(bpy)32 + -silica
Amperometric Amperometric ECL
4–10 ng/mL 0.5–5 pg/mL –
[78] [79] [80]
Ab1/Ag/Ab2-MWCNTs-SiO2
0.1–30.0 ng/mL
[81]
IL-6 CA153 CA125 CA199 CEA IL-8 HCG
Au-Ab1/Ag/Ab2-Poly-HRP Au-Sol-gel-HRP-Ab/Ag/H2O2
Galvanostatic deposition Amperometric Amperometric
0.5 pg/mL 0.5 pg/mL 1 pg/mL 0.25 pg/mL 0.018 ng/mL 10 fg/mL 0.06 U/mL 0.03 U/mL 0.10 U/mL 0.04 ng/mL 1 fg/mL 0.0026 mU/ mL 0.01 ng/mL 10 fg/mL 0.093 pg/mL, 0.061 pg/mL 0.6 pg/mL 1.0 pg/mL
10–1300fg/mL. 0.084–16 U/mL 0.11–13 U/mL 0.16–15 U/mL 0.16–9.2 ng/mL 10–2000 pg/mL 0.005–500 mIU/mL
[82] [83]
0.01–160 ng/mL – 0.1–5.0 ng/mL
[86] [87] [88]
Au-Ab1/Ag/Ab2-Magnetic beads-HRP AuNPs-MWCNTs-Ab1/Ag/Ab2-HRP-Au-thionine-mesoporous nanoparticles MCM-41 Thionine@Au-Ab1/Ag/HRP-Ab2 GSH-AuNP-Ab1/Ag/Ab2-MB-HRP SPE-Ab1/Ag/Ab2-CNT-Ag NPs
Amperometric Amperometric
CNT-Chitosan-AuNPs-Ab1/Ag/Ab2-PtAg@CNCs Fe3O4-Graphene-Ab1/Ag/Ab2-Au hollow NPs
ECL Amperometric
CEA CEA
Ru(bpy)32 + -GO-Nafion-Ab1/Ag/Ab2-SA-QDs Graphene-1-pyrenebutanoic acid, succinimidyl ester-Ab1/Ag/ Ab2-Graphene sheet -Thionine-HRP Graphene-CdS-Ab1/Ag/Ab2-Luminol-AuNP AuNPs@Graphene-Ab1/Ag/Ab2-ZnO NPs@Graphene-GOD
AFP CEA
Au electrode-Thioglycolic acid-AFP/Ab-CdSe Fe3O4/CdSe-CdS-Ab/Ag/K2S2O8
ECL ECL
AFP
Ab-Fe3O4@Au-HRP/Ag/H2O2
Amperometric
CEA PSA CEA AFP PSA CEA AFP CEA PSA
biocompatibility, low toxicity, and a larger specific surface area which can be modified. Due to their magnetic property, magnetic nanoparticles can be attracted by a magnetic field and are easily separable in solution [98,99]. Li et al. [100] reported an electrogenerated chemiluminescence (ECL) immunoassay by using magnetic nanobeads (MNBs) as the carrier. CEA and MNBs were immobilized on a platform via sandwich immunoreaction. The MNBs were released from the platform and labeled with Ru(bpy)32 + species. After the MNBs with Ru(bpy)32 + were immobilized on an Au electrode, ECL was measured for CEA determination. The detection limit of was 1.6 pg/mL. The applications of magnetic particles in the preparing immunosensors were summarized in Table 1.
Amperometric SPR ASV
[84] [85]
[89] [90]
ECL Amperometric
1 pg/mL–50 ng/mL 0.01–80 ng/mL 0.01–200 ng/mL 0.002 pg/mL 0.005–0.5 pg/mL 1 pg/mL 0.002–10 ng/mL
ECL ECL
0.01 ng/mL 3.3 pg/mL
[93] [94]
3–5 ng/mL 10 pg/mL–80 ng/ mL 0.005 mg/L 0.05–100 mg/L 0.032 pg/mL 0.064 pg/mL–10 ng/ mL 5 pg/mL 0.01–200 ng/mL
[91] [92]
[95] [96] [97]
or oxidase enzymes were released. Signal amplification is obtained. For example, Viswanathan [102] developed a disposable electrochemical immunosensor for the detection of CEA in saliva and serum. They covalently immobilized monoclonal anti-CEA antibodies on polyethyleneimine wrapped multiwalled carbon nanotubes screenprinted electrode. The electroactive regents of ferrocene carboxylic acid were encapsulated in liposomes, then a sandwich immunoassay was performed with CEA and CEA tagged liposomes, and numerous ferrocene carboxylic acid molecules were released. The square-wave voltammetry (SWV) was employed to analyze faradic redox responses, the peak current was directly related to the concentration of CEA. The calibration curve for CEA concentration was in the range of 5 10 12 to 5 10 7 g/ mL with a detection limit of 1 10 12 g/mL.
3.4 Controlled Release of Electroactive Regents The technology of the controlled release of electroactive regents is an effective way to improve the detection sensitivity [101]. Liposomes are often used to capsulate the electroactive probe or oxidase enzymes. Antibodies are bound on liposomes. When the analyte antigens were captured by the bound antibody, the liposomes were lysed with surfactant and then numerous electroactive probes 2218
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3.5 Polymers Combination with Functional Groups Polymers with more than one combination of functional groups and binding sites are often used to increase the number of the combination of labels or tags, which enlarge the measured signal in the electrochemical immunosensor [103–105]. In addition, conducting polymers can act as transducers in sensors, which convert a biochemical
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Fig. 4. (A) Multistep synthesis of hybrid gold/SiO2/CdSe-CdS-QD nanostructures. (B) The fabricating steps of the ECL immunosensor. (Adapted from Jie et al. [120])
signal resulting through the interaction of a biological component into an electronic signal. According to the results reported by Jie et al. [106], a sensitive ECL immunosensor could be obtained by the combination of carbon nanotubes (CNTs) and CdSe quantumdots (QDs) in the poly (diallyldimethylammonium chloride) (PDDA) film. PDDA is an ordinary and water-soluble cationic polyelectrolyte. In this paper, PDDA was used to conjugate the CdSe QDs-CNTs composite film on the electrode. By the enrichment of the nanoparticles, the ECL signal was significantly enhanced. Simultaneously, gold nanoparticles (GNPs) were assembled onto the CdSe QDs-CNTs/ PDDA modified electrode, the ECL signals were amplified once again. Human IgG (Ag) was selected as the analyte. Goat anti-human IgG (Ab) was immobilized onto the electrode through GNPs, the ECL signals after the combination reaction were measured. It was asserted that the function of PDDA was firstly explored to develop an ECL biosensor for enhancement of QDs ECL. Electroanalysis 2012, 24, No. 12, 2213 – 2229
4 Electrochemical Sensors for Cancer Biomarkers Detection 4.1 Embryonic Antigen Biomarkers Embryonic antigens are a type of normal components released from embryonic tissue during embryonic development. They reduce in the late embryonic stage and gradually disappear. However, when the cells become cancerous, embryonic antigens can be resynthesized, and expressed largely. Embryonic antigens normally include AFP and CEA, they are the mostly used biomarkers in cancer diagnosis. They are also the analytes which are commonly detected in the assay by electrochemical biosensors [107–110]. Giannetto et al. [111] represented a voltammetric immunosensor for determination of AFP in serum. ELISA assays with voltammetric measurement were carried out that exploited the peculiar properties of nanobiocompo-
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site materials based on gold nanoparticles for the immobilization of Antibody/Antigen/Antibody-HRP sandwich on the glassy carbon electrode (GCE) surface. The electrochemical transduction was mediated by thionin. The limit of quantitation (11 ng/mL) was gained. The stability of the GC/Ab functionalized substrate was demonstrated over one month period. Based on similar analytical principles, Ho et al. [112] developed a sensitive electrochemical immunoassay system for the detection of a protein tumor marker of CEA, that is based on a carbon nanoparticle/poly(ethylene imine)-modified screen-printed graphite electrode (CNPs-PEI/SPGE) covered with anti-CEA antibodies. The signal amplification came from using CdS nanocrystals as biotracers and CNPs as electron transfer enhancer. Square wave anodic stripping voltammetry was performed to record the signal current response obtained from the dissolved CEA-CdS. The calibration curve for CEA concentration was linear in the range of 0.032– 10 ng/mL; and the detection limit was 32 pg/mL. The method is suitably precise and sensitive to function as a means of determining urinary CEA, which is a better marker than serum CEA for the early detection of urothelial carcinoma. Nanomaterials such as graphene oxide and quantum dots were also introduced into the ECL sensor for embryonic antigen determination, due to their excellent properties [113–118]. Guo et al. [119] fabricated an ECL immunosensor for ultrasensitive detection of AFP using graphene-CdS quantumdots-alginate (G-CdS QDs-AL) as the immobilizing support and CdSe/ZnS QDs as the label. CdSe/ZnS QDs could effectively scavenge the ECL of G-CdS QDs-AL composite, and the quenched ECL in-
tensity depended linearly on the logarithm for AFP concentration in the range from 0.05 to 500 fg/mL. The detection limit was 20 ag/mL. Jie et al. [120] reported a sensitive ECL immunosensor for the detection of a protein tumor marker of CEA based on enhanced ECL from a hybrid gold/silica/CdSe-CdS quantum-dot nanostructure. The well-defined hybrid nanostructure comprises a CdSe-CdS QDs core and a dense monolayer of gold NPs separated by a silica shell (Figure 4). The superstructure not only enhanced the ECL intensity by 17 orders of magnitude compared with the pure QDs, but also significantly improved the biocompatibility. Both APS and gold NPs as cross-linkers for an immunosensor could dramatically amplify the ECL signals. This immunosensor showed a detection limit of 64 fg/mL with a wider linear range over 5 orders of magnitude. We also designed a quick and reproducible electrochemical-based immunosensor technique [121] by using magnetic core/shell particles that are coated with a selfassembled multilayer of nanogold. Magnetic particles that are structured from Au/Fe3O4 core-shells were prepared and aminated after a reaction between gold and thiourea, and additional multilayered coatings of gold nanoparticles were assembled on the surface of the core/shell particles. The antibody of anti-CEA was immobilized on the modified magnetic particles, which were then attached on the surface of solid paraffin carbon paste electrode by an external magnetic field (Figure 5). The linear range for the detection of CEA was from 0.005 to 50 ng/mL and the limit of detection (LOD) was 0.001 ng/mL. The LOD is approximately 500 times more sensitive than that of the traditional enzyme-linked immunosorbent assay for CEA
Fig. 5. Scheme for the immobilization of CEA antibody onto SCPE surface and competitive immunoreaction. (Adapted from Li et al. [121])
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detection. The sensor was ready to be regenerated only by renewal of the magnetic nanoparticles. 4.2 Carbohydrate Antigens Biomarkers Carbohydrate antigens, also called cancer antigens, are the variations of the sugar chain, which are mainly produced in cancer cells, but rarely produced in normal tissues or benign lesions of tissues. The carbohydrate antigens commonly detected are CA125, CA15-3, CA19-9, CA549, etc. Among them, CA125 antigen is the biomarker of epithelial ovarian cancer, endometrial cancer, fallopian tube cancer, lung cancer and other tumor markers in a variety of cancers. CA15-3 and CA549 are associated with breast cancer malignancy, which is significant for diagnosis of breast and ovarian cancer. The research of electrochemical sensors on these antigens is also an emerging field [122–125]. Fu [126] developed an electrochemical immunosensor for the detection of CA125 by immobilization CA125 antibody (anti-CA125) on gold hollow microspheres and porous polythionine modified GCE. The gold hollow microspheres provided a biocompatible microenvironment for proteins, and greatly amplified the coverage of antiCA125 molecules on the electrode surface. HRP-labeled anti-CA125 was selected as a signaling molecule, hydroquinone and H2O2 were added in solution. The detection was based on the current change before and after the antigen-antibody interaction the amperometric changes were proportional to CA125 concentration ranging from 4.5 to 36.5 U/mL with a detection limit of 1.3 U/mL. Multibiomarker assays are of great significance in clinical diagnosis. Jia et al. [127] designed a label-free multiple tumor marker parallel detection system based on a light addressable potentiometric sensor. The sensor was constructed of arrayed chips of Si3N4-SiO2-Si which were prepared on silicon wafers. The parallel detection system was developed with controlling interfaces. The l-3,4-dihydroxyphenyl-alanine (l-dopa) hydrochloric solution was used to initiate the surface of the sensor. An l-dopa initiated chip was biofunctionalized respectively by combining it with the antigens and antibodies of four tumor markers, AFP, CEA, CA19-9 and Ferritin. Then unlabeled antibodies and antigens of these four biomarkers were detected. The sensor system is label-free and user friendly. Electrochemiluminescence immunosensor was also widely used to detect the carbohydrate antigen types of cancer markers. We proposed a novel ECL immunoassay based on enzyme amplification, magnetic nanoparticle enrichment and magnetic force-controlled renewal of electrode film [128]. Fe3O4 magnetic nanoparticles loaded with anti-CA125 were attached on the magnetic forcecontrolled GCE then sandwich-type immunoassay was performed via the immunoreactions among glucose oxidase-labeled anti-CA125 and CA125. ECL was generated by the reaction between luminol and hydrogen peroxide. The CA125 concentrations were determined within the range of 0–10 mU/mL, and the detection limit was Electroanalysis 2012, 24, No. 12, 2213 – 2229
8.0 mU/mL. The proposed ECL method also provided a simple selectivity immunoassay protocol, which was applied in the determination of CA125 in clinical serum samples. Wang et al. [129] also prepared an ECL immunosensor for ultrasensitive detection of the tumor biomarker CA125 via a metal-organic nanocomposite with synergistic catalysis function. Silver nanoparticles and nicotinamide adenine dinucleotide hydride (NADH) participated and catalyzed the ECL reaction. The ECL immunosensor was assembled by doping Ru-SiO2 nanoparticles modified electrode with the NADH-AgNPs as immune labels. The chitosan-suspended nanoparticles were cast on the gold electrode surface to immobilize the ECL probes and link gold nanoparticles. The primary antibodies were loaded onto the modified electrode via the gold sulfhydryl covalent binding. The ECL response signal pushed the detection limit down to 0.03 U/mL.
4.3 Enzyme and Isozyme Biomarkers Enzymes are the biological catalysts regulating the metabolism of biological systems and were first used as tumor markers for clinical diagnosis [130]. Isozymes (also known as isoenzymes) are enzymes that catalyze the same chemical reaction but differ in their amino acid sequence, the physical and chemical properties and the immunogenicity. These enzymes usually display different kinetic parameters, for example, Michaelis constant values, or different regulatory properties. In many cases, isozymes (or isoenzymes) are coded by homologous genes that have diverged over time. In general, allozymes precisely represent enzymes from different alleles of the same gene, and isozymes represent the enzymes from different genes that process or catalyze the same reaction. It is often indiscriminate language when the two words are used interchangeably. Isoenzymes are important biomarkers to study carcinoid and carcinoid organizations. The change of serum isoenzymes is often used to diagnose carcinoid tumors [131]. These markers include g-glutamyltranspetidase (gGT), prostatic acid phosphatase (PAP), and lactate dehydrogenase (LDH). The research of these cancer marker focuses on the mechanism of pathogenesis and release. The analysis of the markers was mostly performed by conventional immunoassay and the electrochemical sensors are relatively rarely reported upon. Kelly et al. [132] developed an amperometric immunosensor for the detection of the LDH isoenzyme. Polyclonal antibodies for LDH were covalently immobilized onto a pre-activated ABC membrane, reacted with standard LDH solutions and placed onto a platinum working electrode. Lactate dehydrogenase activity has been measured by detection of the oxidation of NADH at the electrode surface. A calibration curve for LDH in the 0.4– 1.3 U/mL range has been obtained. It provided better selectivity by further using thermal treatment of the membranes.
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Hong et al. [133] designed a screen-printed biosensor for amperometric determination of LDH based on the same dehydrogenase reaction of NAD + /NADH. The printing ink consisted of l-lactate, NAD + , 3,4-dihydroxybenzaldehyde (3,4-DHB) and graphite (acting as the conducting material) was cast on the carbonous working electrode. After the electropolymerization of 3,4-DHB, the sensitive film was formed on the electrode surface. The NADH generated by the LDH could be electro-oxidized and the currents were recorded. The response was linear up to 500 U/L of LDH, and the detection limit was observed in 50 U/L. Prostate specific antigen has been identified as the most reliable pre-treatment tool for diagnosing and monitoring prostate cancer. Liu et al. [134] proposed a strategy for preparing the immunosensor based on the enzymeconjugated prostate-specific antibody (HRP-anti-PSA) reversible binding with a self-assembled phenylboronic acid monolayer on gold. After incubating an HRP-antiPSA electrode in a PSA solution, a decrease in the electrocatalytic response of the HRP-anti-PSA modified electrode to the reduction of H2O2 is observed. The amperometric immunosensor shows a linear increase of the relative intensity in two PSA concentration ranges from 2 to 15 ng/mL and 15 to 120 ng/mL, respectively. Liu et al. [135] had done some research on the detection of prostate cancer-related marker. They developed a disposable electrochemical immunosensor diagnosis device that integrates the immunochromatographic strip technique with an electrochemical immunoassay and ex-
ploits quantum dot (QD) as labels for amplifying signal output. The sandwich immunoreaction was performed on the strip, and the captured QD were dissolved and Cd2 + was determined by stripping voltammetric measurement with a disposable screen-printed electrode. The device takes advantage of the speed and low cost of the conventional immunochromatographic strip test as well as the high sensitivity of the nanoparticle-based electrochemical immunoassay. The device has been successfully applied for the detection of PSA in human serum sample with a detection limit of 20 pg/mL. Morimoto [136] reported a device for the detection of g-GTP enzyme activity in human serum samples by using a freeze-dried substrate-bovine serum albumin (BSA) matrix packed in a micro flow channel. The device consisted of a glass chip with an amperometric l-glutamate sensor and a polydimethylsiloxane (PDMS) sheet with a micro flow channel. g-GTP was used as the test enzyme and the reaction product, l-glutamate was detected using an integrated l-glutamate sensor. The relationship between the slope of the response curve and the enzyme activities was linear in the range of 0–300 U/L for g-GTP. 4.4 Protein Biomarkers The concentration changes of the protein cancer markers are closely related to the cancer occurrence and development, and are directly related to the response to treatment. Therefore, the assay of protein markers is necessary for the development of targeted drugs, early diagnosis
Fig. 6. Multiprotein electrical detection protocol based on different inorganic colloid nanocrystal tracers. (A) Introduction of antibody-modified magnetic beads. (B) Binding of the antigens to the antibodies on the magnetic beads. (C) Capture of the nanocrystallabeled secondary antibodies. (D) Dissolution of nanocrystals and electrochemical stripping detection. (Adapted from Liu et al. [141])
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and treatment of cancer tumors [137]. Protein markers commonly include b2-microglobulin, Bence-Jones protein [138] and they also include a variety of growth factors, such as Epidermal growth factor (EGF) [139] insulin-like growth factor (IGF), etc. [140] Development of the analytical methods for the detection of the cancer markers is very important. However, there were fewer references reported on the application of electrochemical sensors for the assay of protein cancer markers. Liu et al. [141] described an electrochemical immunoassay protocol for the simultaneous measurements of proteins, based on the use of inorganic nanocrystal tracers of ZnS, CdS, PbS, and CuS colloidal crystals. The multiprotein electrical detection capability is coupled with the amplification feature of electrochemical stripping transduction and with an efficient magnetic separation. The multianalyte electrical sandwich immunoassay involves a dual binding event, based on antibodies linked to the nanocrystal tags and magnetic beads. Carbamate linkage is used for conjugating the hydroxyl-terminated nanocrystals with the secondary antibodies (Figure 6). Each biorecognition event yields a distinct voltammetric peak, represented b2-microglobulin, IgG, bovine serum albumin, and C-reactive protein. With the labels of nanocrystal tracers, fmol detection limits were observed. Stoeva [142] has developed a strategy for simultaneously determining three protein cancer markers (PSA, HCG and AFP) at low-femtomolar concentration with the biobarcode assay. They synthesized three Au nanoparticles co-functionalized with barcode strands and a monoclonal or polyclonal antibody that could recognize the target protein, and three magnetic microparticles loaded with monoclonal antibody specific for the target antigen which was correspondingly recognized by the Au NP probe. After the magnetic probes reacted with analytes, the sandwiched reaction of Au NP probe-Ab, target, magnetic microparticles-Ab occurred. The complexes were isolated via magnetic separation, and the barcode strands were released by a ligand exchange process. Finally, the barcode strands were identified by scanometric method. Lai et al. [143] developed an electrochemical, aptamerbased sensor for the detection of platelet-derived growth factor (PDGF) directly in blood serum. The voltammetric current changes derived from target-induced folding in a methylene blue-modified, PDGF-binding aptamer were monitored. They detected the concentration of PDGF at 1 nM directly in undiluted, unmodified blood serum and at 1.25 ng/mL in serum-diluted 2-fold with aqueous buffer. The sensor is reusable and label-free. Proximity ligation assay (PLA) is a new strategy and highly selective and sensitive method for detecting proteins [144]. Zhang [145] developed a novel electrochemical biosensor for a single-step detection of a homodimer protein PDGF-BB based on proximity-dependent surface hybridization assay. This strategy relied on simultaneous recognition of a target molecule by a pair of affinity probes, which was a prerequisite for efficiently promoting the ferrocene-labeled tail sequences of the proximate afElectroanalysis 2012, 24, No. 12, 2213 – 2229
finity probe pair to hybridize together with surface-tethered oligonucleotide, thus triggering the redox current of ferrocene at the electrode. The strategy was a universal methodology for developing high-performance biosensors with nonspecific interferences and reusability.
4.5 Oncogene and Oncogenic Production Any functionally effective change to the genome is by definition reflected in changes in gene expression. Therefore, any mechanism of gene regulation can be involved in genetic changes to epigenetic alterations, since epigenetic features can be modified in order to reverse disease-defining changes and thus facilitate treatment. For example, specific methylation patterns of DNA may be produced by a limited subset of cells undergoing cell death in response to a particular stimulus [146]. DNA is released due to lysis of circulating cancer cells, or micrometastases. It is supposed that metastatic tumor cells can circulate in the blood and circulating DNA can be detected in the plasma of patients in whom circulating metastatic cells are not found [147]. Wei et al. [148] reported an electrochemical detection of salivary mRNA based on specific signal amplification with a hairpin probe for oral cancer mRNA markers. The specificity of hairpin probe (HP) without a linker was tested with cross-detection of two targets. When no target bound to the probe, the hairpin was closed. HRP could not form an effective complex and no signal was observed (Figure 7). After hybridization with the target, the hairpin opened up and the HRP complex was formed. The 3,3’,5,5’-tetramethylbenzidine (TMB) regenerated the reactive HRP, thus amplifying the current signal. Endoge-
Fig. 7. Illustration of the specific signal amplification in hairpin probe electrochemical detection. (Wei et al. [148])
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Fig. 8. Illustration of the multienzyme labeling amplification strategy using HRP-p53392Ab2-GO conjugate. (Adapted from Du et al. [149])
nous Interleukin-8 mRNA is detected in concentration about 400 pM. The activation of oncogenes and the mutation of tumor suppressor genes may cause the normal cells into a malignant transformation, which results in tumorigenesis. Therefore, the expression protein of oncogene can act as a tumor marker. The p53 tumor suppressor gene protein is the most popularly studied cancer gene product. p53 phosphorylation plays an important role in many biological processes and have been used as a cancer biomarker in clinical diagnoses. Du et al. [149] reported an electrochemical immunosensor for ultrasensitive detection of phosphorylated p53 at Ser392 (phospho-p53392) based on graphene oxide as a nanocarrier. HRP and p53392 antibody (p53392Ab2) were linked on the nanoparticles of graphene oxide (HRP-p53392Ab2-graphene oxide). With a sandwich immunoreaction, the HRP-p53392Ab2-graphene oxide was captured onto the electrode surface and the reduction of enzymatically oxidized thionine in the presence of hydrogen peroxide produced an amplified electrocatalytic response (Figure 8). Phosphop53392 could be determined proportionally in the concentration range of 0.02–2 nM with the response current, and the detection limit was 0.01 nM. This immunosensor shows great promise for the detection of other phosphorylated proteins, and it is simple and low-cost for clinical applications. In addition, the p53 gene sequences can also be detected by using the electrochemical sensor. The mutation of tumor suppressor p53 gene is common in malignant tumors. Marquette et al. [150] developed an electrochemical biochip for the detection of DNA sequences of p53. The entire biochip was constructed based on the screen-printing technique and exhibits eight working electrodes that could be individually addressed and grafted through a simple electrochemical procedure. Single-stranded DNA with a C6-NH2 linker at the 5-end was then covalently bound to the surface to act as probe for the direct detection of complementary strands, a particular codon (273) localized in the exon 8 of the p53 gene (20 mer). It enabled the detection of target sequences from 1 to 200 nM. p53 antibodies are products of immunoresponse against abnormal p53 protein. ELISA is the current method for detecting p53 antibodies. However, it requires a long time with multiple steps, and the assay is only semi-quantita2224
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tive. Yan et al. [151] developed a method for quantitative detection of p53 antibodies in human serum using magnetic ECL immunosensor. The immunoassay is based on three antibodies (a biotinylated capture antibody loaded on magnetic microbeads, a detector antibody, and secondary antibody labeled with ruthenium(II) tris-bipyridal) sandwich in which the biotinylated capture antibody banded with the p53 protein. The detector antibody was added to bind the p53 protein at another site. When the secondary antibody labeled with ruthenium(II) tris-bipyridal, was added and bound to the bead-load sandwiched immune-complex, ECL generated in the presence of an excess of tripropylamine. The light was detected and measured. The calibration curve was linear from 0.01 to 1000 ng/mL. The assay has many advantages on sensitivity, linear range, and assay time over the commonly used ELISA method. Protein tyrosine kinases (PTKs) play a central role in human cancers and have emerged as the promising new targets. Clinical studies suggest that overexpression or deregulation of PTKs may be of prognostic or therapeutic value in patients and may indicate an aggressive tumor biology or may predict poor response to therapy and shorter survival. Kerman et al. proposed a label-free electrochemical method for PTKs activity based on peptide tyrosine being electrochemically oxidized [152]. The approach is based on the quantitative detection of peptide tyrosine (Tyr) phosphorylation using the electrochemical oxidation current signal of Tyr. When the phosphorylation is achieved, the phosphorylated Tyr cannot be oxidized at 0.65 V on a multiwalled carbon nanotube-modified screen-printed carbon electrode. When the phosphorylation is successfully inhibited, Tyr can be oxidized and result in a high current response. From the inhibition kinetics, the activity of cellular-sarcoma non-receptor PTK for combination with peptide was determined. 4.6 Hormone Biomarkers Hormones are the regulatory substances secreted by the specific cells in organisms. Some normal tissues which do not produce the hormone under normal circumstances may release peptide hormones (ectopic endocrine hormones) in malignant transformation and lead to the corresponding syndrome. Therefore, the increases of these
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Fig. 9. Schematic display of the reactions and protocols involved in the development of disposable electrochemical immunosensors for ACTH using screen-printed electrodes modified with phenylboronic acid. (Adapted from Guzmn et al. [162])
ectopic endocrine hormones can also be used as a tumorassociated mark. The detection of the hormones by electrochemical analysis is mainly focused on the application of amperometric and ECL sensors [153–156]. Chen et al. [157] constructed an amperometric immunosensor for determination of human serum chorionic gonadotrophin (hCG) by immobilization of hCG with titania sol-gel on a GCE and the direct electrochemistry of HRP labeled to hCG antibody (HRP-anti-hCG). HRPanti-hCG was conjugated with the immobilized hCG, and a direct electron transfer of HRP was observed at the HRP-anti-hCG-hCG/titania sol-gel membrane modified electrode. After a competition between the immobilized HRP-anti-hCG-hCG and the analyte hCG in solution, the current of HRP decreased, 2.5 to 12.5 mIU/mL hCG. Since hCG and two of its specific antibodies can perform electrode reaction in an aqueous solution, the electrochemical protein oxidation signal can be used to determine the hCG by adsorptive stripping voltammetry [158,159]. Kerman et al. [160] reported a rapid immunoassay for the label-free voltammetric detection of hCG in urine based on monitoring the changes in the current signals of anti-hCG before and after the binding of the hCG. The limit of detection of the method was 20 pM (20 mIU/ mL). Chai et al. [161] assembled nano-Au and methylene blue (MB) into films layer-by-layer on GCE modified with thiourea for detection of hCG. The assembled MB acts as the signal tracer. The peak currents decrease after anti-HCG was adsorbed on the nano-Au monolayer because the membrane became less conductive. When the modified immunosensor was incubated in hCG, the peak currents decrease further due to the form of anti-HCG/ HCG immunocomplex, which acts as the inert electron Electroanalysis 2012, 24, No. 12, 2213 – 2229
and mass transfer blocking layer. The working range was 1.0–100.0 mIU/mL with a detection limit of 0.3 mIU/mL. Guzmn et al. [162] reports for the first time an electrochemical immunosensor for the determination of adrenocorticotropic hormone (ACTH). Anti-ACTH antibodies were immobilized onto screen-printed carbon electrode surfaces, a competitive immunoassay between the antigen and the biotinylated hormone for the binding sites of the immobilized antibody was performed (Figure 9). The response was generated by using alkaline phosphatase-labeled streptavidin and 1-naphtyl phosphate as the enzyme substrate, oxidation-currents of the enzyme reaction product, 1-naphtol, was measured by DPV. A detection limit of 18 pg/L was obtained. Liao et al. [163] developed a sandwich-type ECL immunoassay utilizing apoferritin-templated poly(ethylenimine) (PEI) nanoparticles as labels based on the in situ release of the co-reactant of PEI for detection of hCG. The interfusion of graphene and CNTs in Nafion film was coated on the electrode for Ru(bpy)32 + adsorption. Au nanoparticles were assembled to improve the ECL intensity and facilitate the loading of capture hCG antibody. For the sandwich-type immunoreactions, sensitivity could be enhanced by the release of the co-reactant of PEI, which was encapsulated in the protein cage of apoferritin. The minimum detection limit was 0.17 mIU/mL. Kulmala [164,165] and his cooperators developed heterogeneous and homogeneous immunoassays of human thyroid stimulating hormone (hTSH) on an immunometric basis using aromatic Tb(III) chelates as electrochemiluminescent labels and varied types of disposable oxide-covered aluminum electrodes as the solid phase of the immunoassays. The present labels allow for use of time-resolved ECL detection and provide the low detection
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Table 2. Some study related to used HRP-labeled antibody for the detection of carbohydrate antigens. Target Composition of sensitive film
Detection method
Detection limit
Linear range
Ref.
AFP AFP CEA AFP NADH NADH CEA CA125
Potentiometric Amperometric Amperometric ASV Amperometric Amperometric Amperometric Amperometric
0.8 mg/L 5 ng/mL 0.1 ng/mL 9.6 pg/mL 15 mmol/L 5 nmol/L 0.9 ng/mL 0.1 U/mL
2–197 mg/L 15–350 ng/mL 0.5–167 ng/mL 0.02–3.5 ng/mL 0.05–1.83 mmol/L – 1.5–80 ng/mL 0.5–55 U/mL
[167] [168] [169] [170] [171] [172] [122] [123]
Amperometric Amperometric
2–14 U/mL 0.4–140 kU/L 0.5–330 kU/L 0.8–190 kU/L 0.1–44 mg/L 0–30 U/mL 2–30 U/mL
[124] [125]
Amperometric Amperometric
1.29 U/mL 0.2 kU/L 0.5 kU/L 0.3 kU/L 0.1 mg/L 1.73 U/mL 1.37 U/mL
Amperometric Amperometric
0.1 U/mL 3 fg/mL
Gelatin-AgNPs-Ab film/Ag SAMs-AuNPs-Ab1/Ag/Ab2-HRP Thionine/HRP-Ab/Ag Magnetic bead-Ab1/Ag/Au-DNA-CdS-Ab2 Meldolas blue-SWNT-Sol-gel NPs-LDH/NADH AuNPs-thiol-Sol-gel-NADH Fe3O4 NPs-Ab/Ag Glutaradehyde- poly(amidoamine) fourth-generation dendrimer-Fe3O4-Ab/Ag CA125 Sol-gel-HRP-Ab/Ag CA125 Au NPs-Sol-gel-HRP-Ab/Ag CA153 CA199 CEA CA125 Au NPs-Ab1/Ag/Ab2-HRP CA19– Au-HPR-Ab1/Ag 9 CA125 Fe3O4 NPs-Ab1/Ag/Ab2-SiO2-thionine-HRP CEA Phenylenediamine-AuNP-Ab/Ag AFP AFP
Sol-gel-HRP-Ab/Ag ITO-TiO2-CdS-CS-Ab/Ag
TRF CEA AFP PSA
o-Aminobenzenthiol-Au NPs-Ab/Ag o-Aminophenol-Au NP-Ab/Ag Ab1/Ag/Ab2-Au colloid-alkaline phosphatase Graphene-chitosan-Ab1/Ag/Ab2-Au nanorod-GOD
limits. The primary antibody of immunometric immunoassays was coated upon the aluminum oxide surface by physical absorption. Both noncompetitive and competitive immunoassays based on detection of hot electron-induced ECL of the labels were performed. But the accuracy of the assay might be improved due to the unstable aluminum oxide. Chen et al. [166] prepared a reagent-free electrochemical immunosensor of hCG based on colloidal gold nanoparticle/titania sol-gel composite membrane encapsulation of HRP-labeled hCG antibody (HRP-anti-hCG) on a GCE via a vapor deposition method. The formation of immunoconjugate by a simple one-step immunoreaction between hCG in sample solution and the immobilized HRP-anti-hCG introduced a barrier of direct electrical communication of the immobilized HRP on the electrode surface. The hCG analyte could be determined in two linear ranges from 0.5 to 5.0 mIU/mL and 5.0 to 30 mIU/ mL with a detection limit of 0.3 mIU/mL. This composite membrane could be used efficiently for the entrapment of different biomarkers and clinical applications. Some selected references on the application of electrochemical biosensors used in the assay of the key biomarkers were listed in Table 2.
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0.1–450 U/mL 10 fg/mL–100 ng/ mL Amperometric 138 amol/mL 0.02–2.0 ng/mL Photoelectrochemical 40 pg/mL 50 pg/mL–50 ng/ mL Capacitance 80 pg/mL 0.125–100 ng/mL Amperometric 0.1 ng/mL 0.5–20 ng/mL Amperometric 0.8 ng/mL 1.0–500 ng/mL ECL 8 pg/mL 10 pg/mL–8 ng/ mL
[173] [174] [175] [176] [177] [178] [179] [180] [181] [182]
5 Conclusions Electrochemical immunosensors have been attracting more and more attention and application in the cancer marker analysis due to the merits of simple preparation, fast detection, high sensitivity, and portability. It has advantages over other immunosensors, such as enzyme sensors, chemiluminescence immunosensors and other traditional immunosensors, electrochemical immunosensors. Due to its portability, low cost and high sensitivity, it is believed to be an important analytical tool designed to detect the binding event between antibody and antigen without the need for separation and washing steps. It was of particular significance because of the possibility of hyphenation with simple instrumentation as electrochemical systems and combination with high efficiency of enzymes in signal amplification. However, there are many aspects to the electrochemical sensors that need to be further improved for practical applications in analysis, such as the stability, the life-time and the difficulty of renewal of the sensitive film. Nevertheless, researchers are focused on the development of sensor production techniques, improving the quality and sensitivity of the biosensor for biomarkers. In addition, the development of the electrode array[183–185], multichannel and multianalyte detection [186,187] real-time monitoring release of biomarkers [188], and aptamer based or label-free detection [189,190] will likely be the direction of development of electro-
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chemical sensors for the assay of cancer markers. New detection methods such as proximity ligation assay, techniques such as photo-electrochemical detection, and sensitive film materials such as graphene and quantum dot will be more frequently utilized in the assay of cancer biomarkers. Additionally, it can be further developed to be micro and portable in order to meet the different needs. We predict a bright future for electrochemical biosensors for clinical diagnosis and diagnostic screening of cancer because of its superior performance.
Acknowledgements This work was supported by Grants from the National Institutes of Health of the USA (CA089162) and the National Natural Science Foundation of China (No. 21165007).
References [1] N. Majkic´-Singh, J. Med. Biochem. 2011, 30, 186. [2] R. C. Bast, Clin. Chem. 1993, 39, 2444. [3] Biomarkers Definitions Working Group, Clin. Pharmacol. Therapeut. 2001, 69, 89. [4] S. Kumar, A. Mohan, R. Guleria, Biomarkers 2006, 11, 385. [5] E. P. Diamandis, J. Natl. Cancer Inst. 2010, 102, 1462. [6] O. J. Finn, New Engl. J. Med. 2005, 353, 1288. [7] X. J. Wang, J. J. Yu, A. Sreekumar, S. Varambally, R. Shen, D. Giacherio, R. Mehra, J. E. Montie, K. J. Pienta, M. G. Sanda, P. W. Kantoff, M. A. Rubin, J. T. Wei, D. Ghosh, A. M. Chinnaiyan, New Engl. J. Med. 2005, 353, 1224. [8] D. E. Brenner, D. P. Normolle, Cancer Epidemiol. Biomark. Prev. 2007, 16, 1918. [9] R. Frank, R. Hargreaves, Nat. Rev. Drug Discov. 2003, 2, 566. [10] M. Mascini, S. Tombelli, Biomarkers 2008, 13, 637. [11] J. Wang, Y. Cao, Y. Y. Xu, G. X. Li, Biosens. Bioelectron. 2009, 25, 532. [12] K. T. Yong, H. Ding, I. Roy, W. C. Law, E. J. Bergey, A. Maitra, P. N. Prasad, ACS Nano 2009, 3, 502. [13] X. Y. Wu, H. J. Liu, J. Q. Liu, K. N. Haley, J. A. Treadway, J. P. Larson, N. F. Ge, F. Peale, M. P. Bruchez, Nat. Biotechnol. 2002, 21, 41. [14] X. Wang, Q. Y. Zhang, Z. J. Li, X. T. Ying, J. M. Lin, Clin. Chim. Acta 2008, 393, 90. [15] Z. F. Fu, F. Yan, H. Liu, Z. J. Yang, H. X. Ju, Biosens. Bioelectron. 2008, 23, 1063. [16] S. Prabhulkar, S. Alwarappan, G. D. Liu, C. Z. Li, Biosens. Bioelectron. 2009, 24, 3524. [17] A. C. Barton, F. Davis, S. P. J. Higson, Anal. Chem. 2008, 16, 9411. [18] D. P. Tang, R. Yuan, Y. Q. Chai, Anal. Chem. 2008, 80, 1582. [19] D. W. Chan, R. A. Beveridge, H. Muss, H. A. Fritsche, G. Hortobagyi, R. Theriault, D. Kiang, B. J. Kennedy, M. Evelegh, J. Clin. Oncol. 1997, 15, 2322. [20] W. C. Yang, M. Yu, X. H. A. T. Sun, Lab Chip 2010, 10, 2527. [21] J. Wang, Biosens. Bioelectron. 2006, 21, 1887. [22] B. V. Chikkaveeraiah, A. A. Bhirde, N. Y. Morgan, H. S. Eden, X. Y. Chen, ACS Nano 2012, 6, 6546. Electroanalysis 2012, 24, No. 12, 2213 – 2229
[23] A. Qureshi, Y. Gurbuz, J. H. Niazi, Sens. Actuators B 2012, 171–172, 62. [24] J. F. Rusling, C. V. Kumar, J. S Gutkind, V. Patel, Analyst 2010, 135, 2496. [25] D. R. Thvenota, K. Toth, R. A. Durstc, G. S. Wilson, Biosens. Bioelectron. 2001, 16, 121. [26] P. Skldal, Electroanalysis 1997, 9, 737. [27] I. E. Tothill, Stem Cells Dev. Biol. 2009, 20, 55. [28] M. Perfzou, A. Turner, A. MerkoÅi, Chem. Soc. Rev. 2012, 41, 2606. [29] M. Aizawa, A. Morioka, S. Suzuki, Anal. Chim. Acta 1980, 115, 61. [30] O. A. Sadik, A. O. Aluoch, A. L. Zhou, Biosens. Bioelectron. 2009, 24, 2749. [31] J. Wang, Biosens. Bioelectron. 2006, 21, 1887. [32] J. Tang, J. X. Huang, B. L. Su, H. F. Chen, D. P. Tang, Biochem. Eng. J. 2011, 53, 223. [33] Y. T. Wang, Z. Q. Zhang, V. Jain, J. J. Yi, S. Muellere, J. Sokolova, Z. X. Liu, K. Levong, B. Rigasb, M. H. Rafailovich, Sens. Actuators B 2010, 146, 381. [34] Y. F. Jia, M. Qin, H. K. Zhang, W. C. Niu, X. Li, L. K. Wang, X. Li, Y. P. Bai, Y. J. Cao, X. Z. Feng, Biosens. Bioelectron. 2007, 22, 3261. [35] X. Yu, B. Munge, V. Patel, G. Jensen, A. Bhirde, J. D. Gong, S. N. Kim, J. Gillespie, J. S. Gutkind, F. Papadimitrakopoulos, J. F. Rusling, J. Am. Chem. Soc. 2006, 128, 11199. [36] X. L. He, R. Yuan, Y. Q. Chai, Y. T. Shi, J. Biochem. Biophys. Meth. 2008, 70, 823. [37] N. P. Sardesai, J. C. Barron, J. F. Rusling, Anal. Chem. 2011, 83, 6698. [38] X. Liu, Y. Y. Zhang, J. P. Lei, Y. D. Xue, L. X. Cheng, H. X. Ju, Anal. Chem. 2010. 82, 7351. [39] Y. Zou, Z. Guo, Med. Eng. Phys. 2003, 25, 79. [40] S. Carrara, V. Bhalla, C. Stagni, L. Benini, A. Ferretti, F. Valle, A. Gallotta, B. Ricc, B. Samor , Sens. Actuators B 2009, 136, 163. [41] J. Joo, D. Kwon, C. Yim, S. Jeon, ACS Nano 2012, 6, 4375. [42] G. Y. Shen, M. H. Liu, X. W. Cai, J. L. Lu, Anal. Chim. Acta 2008, 630, 75. [43] Y. Uludag, I. E. Tothill, Anal. Chem. 2012, 84, 5898. [44] L. Mao, R. Yuan, Y. Q. Chai, Y. Zhuo, X. Yang, Sens. Actuators B 2010, 149, 226. [45] N. Sardesai, S. M. Pan, J. Rusling, Chem. Commun. 2009, 33, 4968. [46] P. Sarkar, P. S. Pal, D. Ghosh, S. J. Setford, I. E. Tothill, Int. J. Pharm. 2002, 238, 1. [47] W. J. Miao, A. J. Bard, Anal Chem. 2003, 75, 5825. [48] Y. Liu, H. Jiang, Electroanalysis 2006, 18, 1007. [49] Y. X. Hou, C. Tlili, N. Jaffrezic-Renault, A. D. Zhang, C. Martelet, L. Ponsonnet, A. Errachid, J. Samitierd, J. Bausells, Biosens. Bioelectron. 2004, 20, 1126. [50] M. Camp s, M. G. Olteanu, J. L. Marty, Sens. Actuators B 2008, 1299, 263. [51] S. Dhawan, Peptides 2002, 23, 2091. [52] J. Wang, Analyst 2005, 130, 421. [53] S. Campuzano, J. Wang, Electroanalysis 2007, 19, 769. [54] H. X. Ju, X. J. Zhang, J. Wang, in: Biological and Medical Physics, Biomedical Engineering, Springer, Heidelberg 2011, ch. 2, 39 – 84. [55] M. J. A. Shiddiky, S. Rauf, P. H. Kithva, M. Trau, Biosens. Bioelectron. 2012, 35, 251. [56] J. Wang, Electroanalysis 2011, 23, 1289. [57] M. Pumera, S. Sanchez, I. Ichinose, J. Tang, Sens. Actuators B 2007, 123, 1195.
2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.electroanalysis.wiley-vch.de
2227
Review
J. Li et al.
[58] A. A. Bhirde, V. Patel, J. Gavard, G. F. Zhang, A. A. Sousa, A. Masedunskas, R. D. Leapman, R. Weigert, J. S. Gutkind, J. F. Rusling, ACS Nano 2009, 3, 307. [59] B. V. Chikkaveeraiah, A. Bhirde, R. Malhotra, V. Patel, J. S. Gutkind, J. F. Rusling, Anal. Chem. 2009, 81, 9129. [60] N. P. Sardesai, J. C. Barron, J. F. Rusling, Anal. Chem. 2011, 83, 6698. [61] X. Yu, B. Munge, V. Pate, G. Jensen, A. Bhirde, J. D. Gong, S. N. Kim, J. Gillespie, J. S. Gutkind, F. Papadimitrakopoulos, J. F. Rusling, J. Am. Chem. Soc. 2006, 128, 11199. [62] J. M. Pingarrn, P. YÇez-SedeÇo, A. Gonzlez-Corts, Electrochim. Acta 2008, 53, 5848. . [63] K. Saha, S. S. Agasti, C. Kim, X. N. Li, V. M. Rotello, Chem. Rev. 20121, 12, 2739. [64] F. Lu, T. Doane, J. J. Zhu, C. Burda, Inorg. Chim. Acta 2012, doi.org/10.1016/j.ica.2012.05.038, in press. [65] C. F. Ou, R. Yuan, Y. Q. Chai, M. Y. Tang, R. Chai, X. L. He, Anal. Chim. Acta 2007, 603, 205. [66] W. Yang, K. R. Ratinac, S. P. Ringer, P. Thordarson, J. J Gooding, F. Braet, Angew. Chem. Int. Ed. 2010, 49, 2114. [67] M. Pumera, A. Ambrosi, A. Bonanni, E. L. K. Chng, H. L. Poh, Tr. Anal. Chem. 2010, 29, 954. [68] R. Chowdhury, S. Adhikari, P. Rees, S. P. Wilks, F. Scarpa, Phys. Rev. B 2011, 83, 045401. [69] D. J. Lin, J. Wu, M. Wang, F. Yan, H. X. Ju, Anal. Chem. 2012, 84, 3662. [70] Z. P. Chen, Z. Feng, J. H. Jiang, X. B. Zhang, G. L. Shen, R. Q. Yu, Sens. Actuators B 2008, 129, 146. [71] J. J. Zhang, Y. Liu, L. H. Hu, L. P. Jiang, J. J. Zhu, Chem. Commun. 2011, 47, 6551. [72] N. P. Sardesai, J. C. Barron, J. F. Rusling, Anal. Chem. 2011, 83, 6698. [73] J. Zhang, J. P. Lei, C. L. Xu, L. Ding, H. X. Ju, Anal. Chem. 2010, 82, 1117. [74] L. Ding, R. C. Qian, Y. D. Xue, W. Cheng, H. X. Ju, Anal. Chem. 2010, 82, 5804. [75] W. Cheng, F. Yan, L. Ding, H. X. Ju, Y. B. Yin, Anal. Chem. 2010, 82, 3337. [76] G. S. Lai, F. Yan, J. Wu, C. Leng, H. X. Ju, Anal. Chem. 2011, 83, 2726. [77] G. S. Lai, F. Yan, H. X. Ju, Anal. Chem. 2009, 81, 9730. [78] V. Mani, B. V. Chikkaveeraiah, V. Patel, J. S. Gutkind, J. F. Rusling, ACS Nano 2009, 3, 585. [79] R. Malhotra, V. Patel, J. P. Vaque, J. S. Gutkind, J. F. Rusling, Anal. Chem. 2010, 82, 3118. [80] N. P. Sardesai, J. C. Barron, J. F. Rusling, Anal. Chem. 2011, 83, 6698. [81] R. P. Liang, Z. X. Wang, L. Zhang, J. D. Qiu, Sens. Actuators B 2012, 166–167, 569. [82] C .K. Tang, A. Vaze, J. F. Rusling, Lab Chip 2012, 12, 281. [83] J. Wu, Y. T. Yan, F. Yan, H. X. Ju, Anal. Chem. 2008, 80, 6072. [84] B. S. Munge, A. L. Coffey, J. M. Doucette, B. K. Somba, R. Malhotra, V. Patel, J. S Gutkind, J. F. Rusling, Angew. Chem. Int. Ed. 2011, 50, 7915. [85] J. J. Lu, S. Q. Liu, S. G. Ge, M. Yan, J. H. Yu, X. T. Hu, Biosens. Bioelectron. 2012, 33, 29. [86] D. P. Tang, R. Yuan, Y. Q. Chai, Anal. Chem. 2008, 80, 1582. [87] B. S. Munge, A. L. Coffey, J. M. Doucette, B. K. Somba, R. Malhotr, V. Patel, J. S. Gutkind, J. F. Rusling, Angew. Chem. 2011, 123, 8061. [88] G. S. Lai, J. Wu, H. X. Ju, F. Yan, Adv. Funct. Mater. 2011, 21, 2938. [89] M. Zhang, W. J. Dai, M. Yan, S. G. Ge, J. H. Yu, X. R. Song, W. Xu, Analyst 2012, 137, 2112.
2228
www.electroanalysis.wiley-vch.de
[90] J. Tang, D. P. Tang, R. Niessner, G. N. Chen, D. Knopp, Anal. Chem. 2011, 83, 5407. [91] T. Hao, Z. Guo, S. Du, L. Shi, Sens. Actuators B 2012, 171– 172, 803. [92] M. H. Yang, A. Javadi, H. Li, S. Q. Gong, Biosens. Bioelectron. 2010, 26, 560. [93] W. W. Tu, W. J. Wang, J. P. Lei, S. Y. Deng, H. X. Ju, Chem. Commun. 2012, 48, 6535. [94] Y. F. Cheng, R. Yuan, Y. Q. Chai, H. Niu, Y. L. Cao, H. J. Liu, L. J. Bai, Y. L. Yuan, Anal. Chim. Acta 2012, 745, 137. [95] X. L. Fang, M. Han, G. F. Lu, W. W. Tu, Z. H. Dai, Sens. Actuators B 2012, 168, 271. [96] G. Jie, L. Wang, S. Zhan, Chem.-Eur. J. 2011, 17, 64. [97] H. J. Jin, N. Gan, J. G. Hou, F. T. Hu, Y. T. Cao, L. Zheng, Z. Y. Guo, Sens. Lett. 2012, 10, 886. [98] V. Mani, B. V. Chikkaveeraiah, J. F. Rusling, Expert Opin. Med. Diagn. 2011, 5, 381. [99] J. Llandro, J. J. Palfreyman, A. Ionescu, C. H. W. Barnes, MBEC 2010, 48, 977. [100] M. Y. Li, Y. M. Sun, L. Chen, L. Li, G. Z. Zou, X. L. Zhang, W. R. Jin, Electroanalysis 2010, 22, 333. [101] B. Qu, L. Guo, X. Chu, D. H. Wu, G. L. Shen, R. Q. Yu, Anal. Chim. Acta 2010, 663, 147. [102] S. Viswanathan, C. Rani, A. V. Anand, J. A. Ho, Biosens. Bioelectron. 2009, 24, 1984. [103] M. A. Bangar, D. J. Shirale, W. Chen, N. V. Myung, A. Mulchandani, Anal. Chem. 2009, 81, 2168. [104] H. B. Fredj, S. Helali, Z. Sassi, N. Jaffrezic-Renault, A. Abdelghani, Sens. Lett. 2011, 9, 2291. [105] B. D. Malhotraa, A. Chaubeyb, S. P. Singh, Anal. Chim. Acta 2006, 578, 59. [106] G. Jie, L. Li, C. Chen, J. Xuan, J. J. Zhu, Biosens. Bioelectron. 2009, 24, 3352. [107] M. S. Wilson, Anal. Chem. 2005, 77, 1496. [108] J. H. Lin, H. X. Ju, Biosens. Bioelectron. 2005, 20, 1461. [109] K. Kojima, A. Hiratsuka, H. Suzuki, K. Yano, K. Ikebukuro, I. Karube, Anal. Chem. 2003, 75, 1116. [110] W. Q. Lai, J. Y. Zhuang, J. Tang, G. N. Chen, D. P. Tang, Microchim. Acta 2012, 178, 357. [111] M. Giannetto, L. Elviri, M. Careri, A. Mangia, G. Mori, Biosens. Bioelectron. 2011, 26, 2232. [112] J. A. Ho, Y. C. Lin, L. S. Wang, K. C. Hwang, P. T. Chou, Anal. Chem. 2009, 81, 1340. [113] J. Tang, D. P. Tang, R. Niessner, G. N. Chen, D. Knopp, Anal. Chem. 2011, 83, 5407. [114] B. L. Su, J. Tang, J. X. Huang, H. H. Yang, B. Qiu, G. N. Chen, D. P. Tang, Electroanalysis 2010, 22, 2720. [115] Q. Wei, K. X. Mao, D. Wu, Y. X. Dai, J. Yang, B. Du, M. H. Yang, H. Li, Sens. Actuators. B 2010, 149, 314. [116] X. L. Fang, M. Han, G. F. Lu, W. W. Tu, Z. H. Dai, Sens. Actuators B 2012, 168, 271. [117] S. Y. Deng, Z. T. Hou, J. P. Lei, D. J. Lin, Z. Hu, F. Yan, H. X. Ju, Chem. Commun. 2011, 47, 12107. [118] F. Wang, S. S. Hu, Microchim. Acta 2009, 165, 1. [119] Z. Y. Guo, T. T. Hao, S. Wang, N. Gan, X. Li, D. Y. Wei, Electrochem. Commun. 2012, 14, 13. [120] G. F. Jie, P. Liu, S. S. Zhang, Chem. Commun. 2010, 46, 1323. [121] J. P. Li, H. L. Gao, Z. Q. Chen, X. P. Wei, C. F. Yang, Anal. Chim. Acta 2010, 665, 98. [122] X. H. Fu, J. Y. Wang, N. Li, L. Wang, L. Pu, Microchim. Acta 2009, 165, 437. [123] X. H. Fu, Anal. Lett. 2010, 43, 455. [124] Z. Dai, F. Yan, J. Chen, H. G. Ju, Anal. Chem. 2003, 75, 5429. [125] J. Wu, F. Yan, X. Q. Zhang, Y. T. Yan, J. H. Tang, H. X. Ju, Clin. Chem. 2008, 54, 1481. [126] X. H. Fu, Electroanalysis 2007, 19, 1831.
2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Electroanalysis 2012, 24, No. 12, 2213 – 2229
Electrochemical Biosensors for Cancer Biomarker Detection [127] Y. F. Jia, C. Y. Gao, J. He, D. Feng, K. L. Xing, M. Wu, Y. Liu, W. S. Cai, X. Z. Feng, Analyst 2012, 137, 3806. [128] Q. Xu, J. P. Li, S. H. Li, H. C. Pan, J. Solid State Electrochem. 2012, DOI: 10. 1007/s10008-012-1719-2, in press. [129] G. J. Wang, F. Jin, N. Dai, Z. Y. Zhong, Y. Qing, M. X. Li, R. Yuan, D. Wang, Anal. Biochem. 2012, 422, 7. [130] J. A. Ludwig, J. N. Weinstein, Nat. Rev. Cancer 2005, 5, 845. [131] W. H. Fishman, N. I. Inglis, L. L. Stolbach, M. J. Krant, Cancer Res. 1968, 28, 150. [132] S. Kelly, D. Compagnone, G. Guilbault, Biosens. Bioelectron. 1998, 13, 173. [133] M. Y. Hong, J. Y. Chang, H. C. Yoon, H. S. Kim, Biosens. Bioelectron. 2002, 17, 13. [134] S. Q. Liu, X. T. Zhang, Y. F. Wu, Y. F. Tu, L. He, Clin. Chim. Acta 2008, 395, 51. [135] G. D. Liu, Y. Y. Lin, J. Wang, H. Wu, C. M. Wai, Y. H. Lin, Anal. Chem. 2007, 79, 7644. [136] K. Morimoto, S. Upadhyay, T. Higashiyama, N. Ohgami, H. Kusakabe, J. Fukuda, H. Suzuki, Sens. Actuators B 2007, 124, 477. [137] P. Maurya, P. Meleady, P. Dowling, M. Clynes, Anticancer Res. 2007, 27, 1247. [138] M. V. Dhodapkar, G. Merlini, A. Solomon, Hematol./ Oncol. Clin. North Am. 1997, 11, 89. [139] S. V. Sharma, D. W. Bell, J. Settleman, D. A. Haber, Nat. Rev. Cancer 2007, 7, 169. [140] D. LeRoith, C. T. Roberts, Cancer Lett. 2003, 195, 127. [141] G. D. Liu, J. Wang, J. Kim, M. R. Jan, Anal. Chem. 2004, 76, 7126. [142] S. I. Stoeva, J. S. Lee, J. E. Smith, S. T. Rosen, C. A. Mirkin, J. Am. Chem. Soc. 2006, 128, 8378. [143] R. Y. Lai, K. W. Plaxco, A. J. Heeger, Anal. Chem. 2007, 79, 229. [144] O. Sçderberg, M. Gullberg, M. Jarvius, K. Ridderstr le, K. J. Leuchowius, J. Jarvius, K. Wester, P. Hydbring, F. Bahram, L. G. Larsson, U. Landegren, Nature Meth. 2006, 3, 995. [145] Y. L. Zhang, Y. Huang, J. H. Jiang, G. L. Shen, R. Q. Yu, J. Am. Chem. Soc. 2007, 129, 15448. [146] V. V. Levenson, A. A. Melnikov, Pharmaceuticals 2012, 5, 94. [147] R. E. Board, L. Knight, A. Greystoke, F. H. Blackhall, A. Hughes, C. Dive, M. Ranson, Biomark. Insights 2007, 2, 307. [148] F. Wei, J. H. Wang, W. Liao, B. G. Zimmermann, D. T. Wong, C. M. Ho, Nucl. Acids Res. 2008, 36, e65. [149] D. Du, L. M. Wang, Y. Y. Shao, J. Wang, M. H. Engelhard, Y. H. Lin, Anal. Chem. 2011, 83, 746. [150] C. A. Marquette, M. F. Lawrence, L. J. Blum, Anal. Chem. 2006, 78, 959. [151] G. H. Yan, D. Xing, S. C. Tan, Q. Chen, J. Immunol. Meth. 2004, 288, 47. [152] K. Kerman, M. Vestergaard, E. Tamiya, Anal. Chem. 2007, 9, 6881. [153] V. Mani, B. V. Chikkaveeraiah, J. F. Rusling, Expert Opin. Med. Diagn. 2011, 5, 381. [154] M. U. Ahmed, M. M. Hossain, E. Tamiya, Electroanalysis 2008, 20, 616. [155] M. Moreno-Guzmn, A. Gonzlez-Corts, P. YÇezSedeÇo, J. M. Pingarrn, Electroanalysis 2012, 24, 1100. [156] S. Snchez, M. Roldn, S. Prez, E. F bregas, Anal. Chem. 2008, 80, 6508. [157] J. Chen, F. Yan, Z. Dai, H. X. Ju, Biosens. Bioelectron. 2005, 21, 330. [158] K. R. Wehmeyer, H. B. Halsall, W. R. Heineman, Clin. Chem. 1982, 28, 1968. Electroanalysis 2012, 24, No. 12, 2213 – 2229
[159] J. Rodriguez-Flores, R. OKennedy, M. R. Smyth, Biosensors 1989, 4, 1. [160] K. Kerman, N. Nagatani, M. Chikae, T. Yuhi, Y. Takamura, E. Tamiya, Anal. Chem. 2006, 78, 5612. [161] R. Chai, R. Yuan, Y. Q. Chai, C. F. Ou, S. R. Cao, X. L. Li, Talanta 2008, 74, 1330. [162] M. M. Guzmn, I. Ojeda, R. Villalonga, A. GonzlezCorts, P. YÇez-SedeÇo, J. M. Pingarrn, Biosens. Bioelectron. 2012, 35, 82. [163] N. Liao, Y. Zhuo, Y. Q. Chai, Y. Xiang, Y. L. Cao, R. Yuan, J. Han, Chem. Commun. 2012, 48, 7610. [164] S. Kulmala, M. H kansson, A. M. Spehar, A. Nyman, J. Kankare, K. Loikas, T. Ala-Kleme, J. Eskola, Anal. Chim. Acta 2002, 458, 271. [165] J. Eskola, P. M kinen, L. Oksa, K Loikas, M. Nauma, Q. H. Jiang, M. H kansson, J. Suomi, S. Kulmala, Luminescence 2006, 18, 238. [166] J. Chen, J. H. Tang, F. Yan, H. X. Ju, Biomaterial 2006, 27, 2313. [167] Z. Qiang, R. Yuan, Y. Q. Chai, N. Wang, Y. Zhuo, Y. Zhang, X. L. Li, Electrochim. Acta 2006, 51, 3763. [168] Y. Y. Xu, C. Bian, S. F. Chen, S. H. Xia, Anal. Chim. Acta 2006, 561, 48. [169] Z. Dai, F. Yan, H. Yu, X. Y. Hu, H. X. Ju, J. Immunol. Meth. 2004, 287, 13. [170] C. F. Ding, Q. Zhang, S. S. Zhang, Biosens. Bioelectron. 2009, 24, 2434. [171] A. Arvinte, A. M. Sesay, V. Virtanen, C. Bala, Electroanalysis 2008, 20, 2355. [172] B. K. Jena, C. R. Raj, Anal. Chem. 2006, 78, 6332. [173] L. N. Wu, J. Chen, D. Du, H. X. Ju, Electrochim. Acta 2006, 51, 1208. [174] D. Du, X. X. Xu, S. F. Wang, A. D. Zhang, Talanta 2007, 71, 1257. [175] D. P. Tang, B. L. Su, J. Tang, J. J. Ren, G. N. Chen, Anal. Chem. 2010, 82, 1527. [176] X. B. Sun, Z. F. Ma, Biosens. Bioelectron. 2012, 35, 470. [177] F. Yan, J. Wu, F. Tan, Y. T. Yan, H. X. Ju, Anal. Chim. Acta 2009, 644, 36. [178] G. L. Wang, J. J. Xu, H. Y. Chen, S. Z. Fu, Biosens. Bioelectron. 2009, 25, 791. [179] S. Q. Hua, Z. M. Xie, C. X. Lei, G. L. Shen, R. Q. Yu, Sens. Actuators B 2005, 106, 641. [180] H. Tang, J. H. Chen, L. H. Nie, Y. F. Kuang, S. Z. Yao, Biosens. Bioelectron. 2007, 22, 1061. [181] X. P. Liu, H. W. Wu, Y. Zheng, Z. H. Wu, J. H. Jiang, G. L. Shen, R. Q. Yu, Electroanalysis 2010, 22, 244. [182] S. J. Xu, Y. Liu, T. H Wang, J. H. Li, Anal. Chem. 2011, 83, 3817. [183] R. Malhotra, V. Patel, B. V. Chikkaveeraiah, B. S. Munge, S. C. Cheong, R. B. Zain, M. T. Abraham, D. K. Dey, J. S. Gutkind, J. F. Rusling, Anal. Chem. 2012, 84, 6249. [184] B. V. Chikkaveeraiah, V. Mani, V. Patel, J. S. Gutkind, J. F. Rusling, Biosens. Bioelectron. 2011, 26, 4477. [185] G. C. Jensen, C. E. Krause, G. A. Sotzing, J. F. Rusling, Phys. Chem. Chem. Phys. 2011, 13, 4888. [186] H. J. Lee, S. H. Lee, T. Yasukawa, J. Ramn-Azcn, F. Mizutani, K. Ino, H. Shiku, T. Matsue, Talanta 2010, 81, 657. [187] C. K. Tang, A. Vaze, J. F. Rusling, Lab Chip 2012, 12, 281. [188] E. Khazanov, E. Yavin, A. Pascal, A. Nissan, Y. Kohl, M. Reimann-Zawadzki, A. Rubinstein, Pharm. Res. 2012, 29, 983. [189] A. Ilyas, W. Asghar, P. B. Allen, H. Duhon, A. D. Ellington, S. M. Iqbal, Nanotechnology 2012, 23, 275502 [190] Q. Wei, Y. Zhao, C. Xu, D. Wu, Y. Cai, J. He, H Li, B. Du, M. Yang, Biosens. Bioelectron. 2011, 26, 3714.
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