ng/mL in only 10 μL of serum, a mass detection limit 75 times lower than current commercial techniques. This highly sensitive, low cost and fast detection method ...
Single-Walled Carbon-Nanotube Forest Immunosensor for Amplified Detection of Cancer Biomarkers Joseph D. Gonga, Gary Jensena, Ashwin Bhirdea, Xin Yua, Bernard Mungea,c, Voymesh Pateld, SangNyon Kime, J. Silvio Gutkindd, Fotios Papadimitrakopoulose and James F. Ruslinga,b a
Department of Chemistry, 55 N. Eagleville Rd., University of Connecticut, Storrs, CT 06269 Department of Pharmacology, University of Connecticut Health Center, Farmington, CT 06032, c Department of Chemistry, Salve Regina University, Newport RI 02840 d Oral and Pharyngeal Cancer Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland e Institute of Material Science, University of Connecticut, Storrs, CT06269 b
Abstract Prostate Specific Antigen (PSA) is a major biomarker used clinically in the detection of prostate cancer. Current commercial immunoassays for detection of this biomarker rely on spectroscopic absorbance and electrochemiluminescence which are able to detect as low as 0.03 ng/mL of PSA within ȝ/ RI ser u m. I n t hi s p ap e r a no ve l a mp li fied electrochemical technique for detection of Prostate specific antigen will be described. This electrochemical approach is a highly sensitive method that can have detection limit of 0.004 ng/mL in onl\ȝ/RIVHUXPDPDVVGHWHFWLRQ limit 75 times lower than current commercial techniques. This highly sensitive, low cost and fast detection method not only allows for detection of cancer biomarkers in serum, but also in tissue and cells. Moreover, immunoassay arrays based on this methodology will be highly promising for application in large-scale clinical screening and point-o f-care diagnostics. Characterization of the anti-PSA amplification tag in this amplification system using TEM, AFM, SEM, and CE will also be described.
1.Introduction Detection and quantization of proteins and their binding partners are critical for the progress of biomedical research. Modern applications include medical diagnostics, elucidation of d isease vecto rs, immu no lo gy, ne w d rug development and emerging fields such as
p r o t eo mi c s a n d s y s t e m s b i o l o g y [ 1 -4 ] . Measure me nt s of collections of protein biomarkers via such immunological approaches are promising for reliable early caner detection [5-8]. Previous work has shown that usage of chemically active single walled nanotube (SWNT) forests on pyrolytic graphite (PG) electrodes provides excellent electrical links to conductive surfaces [10] and protein immunosensor capabilities [11]. We had demonstrated detection of for Prostate Specific Antigen (PSA) in serum using an electrochemical “sandwich” immunoassay that has the same order of magnitude of detection limit in serum (0.4 ng/mL) as commonly used immunoassay called ELISA. In this work, we will demonstrate a much reduced detection limit of this electrochemical immunoassay b y significantly increasing the ratio of enzyme (HRP) labels to the secondary antibody (Ab2) by conjugating them to highly functionalized multi wa l l e d c a r b o n n a n o t u b e s ( M W C N T ’S ) . Highly functionalized MWCNTs have much more free carboxylate groups around their surface than a single Ab2. Coupling Ab2 and enzyme labels to MWCNTs simultaneously can create more enzyme (HRP) labels per Ab2, which in turn generate much larger electrochemical signal with the Antigen-Ab2 binding event and enable detection limits as low as ~0.004ng/mL (0.1 Fmol/mL).
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The full characterization for the quantitative ratio of HRP labels to Ab2 is determined by Capillary Electrophoresis (CE). Comparison of the dimension of MWCNTs before and after enzyme and Ab2 conjugation using Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) showed a increase of the average width of MWCNTs from 19 nm to 26 nm, indicating the existence of a thin protein film around the CNTs.
2.Experimental 2.1 Materials and Setup Bovine Serum Albumin (BSA), and Horseradish peroxidase (HRP) was purchased from SigmaAldrich. Multi-walled carbon nanotubes where obtained from Helix Material Solutions. PSA standard in serum, monoclonal primary and secondary anti-PSA antibody, and HRPconjugated monoclonal anti-PSA antibody were purchased from Amogen. Recombinant PSA standard in buffer solution was received from Dr. Voymesh Patel at NIH. BSA and primary anti-PSA antibody (Ab1) was dissolved in pH 7.0 phosphate (PBS) buffer with 0.05% Tween-20 (0.01 M in phosphate, 0.14M NaCl, 2.7 mM KCl) prior to use. PSA buffer standard was diluted in pH 7.0 PBS buffer and PSA serum standard were diluted in serum diluents to desired concentration. Secondary anti-PSA antibody (Ab2) and HRP were diluted in pH 7.0 PBS buffer with 0.05% Tween-20 prior to conjugation. 1-(3-(dimethylamino)propyl)-3ethylcarbodiimide hydrochloride (EDAC) and Nhydroxysulfosuccinimide (NHSS) were dissolved in water immediately before use. Single-wall carbon nanotubes (HiPco) were obtained from Carbon Nanotechnologies, Inc and functionalized following established protocol [10]. Fabrication of the “tag” using MWCNT’s , HRP and Ab2 is accomplished in the following steps. First, MWCNTs were sonicated within a mixture of 3:1 sulfuric acid and nitric acid for 6 hrs. The acid treated MWCNT’s were then filtered until pH of supernatant was 6~7. The MWCNT’s were then dried in a vacuum oven at room temperature for 24hrs. 0.5 mg of these
MWCNT’s was dispersed in 1mL of pH 7.0 PBS buffer to make the final concentration 0.5 mg/mL. A mixture of EDC : NHSS (400mM:100mM) was prepared and mixed with MWCNT’s for 20 minutes. The mixture was then centrifuged at 3000 RPM and supernatant discarded. 1mg/mL of HRP solution was added to the remaining activated MWCNTs and quickly re-dispersed using Vortex. Secondary Anti-PSA antibody (Ab2) was then injected into the mixture to a final concentration of 5Pg/ml. The solution was stirred overnight and was ultracentrifuged at 15,000 RPM at 4°C for 40 minutes. Supernatant was removed and the product was mixed with PBS with 0.05% Tween 20. Ultracentrifuging step was then repeated at 3000 RPM for 3 times for washing purpose to remove any loosely bound and unbound HRP and Ab2. The final product was then separated into aliquots and stored at 4°C. The above fabricated Ab2 “tag” was then used in the sandwich assays formed on PG-SWNT forest electrodes fabricated using established protocols [10-11]. First, 30 μL freshly prepared 400 mM EDC and 100 mM NHSS in water were placed onto the SWNT forests and washed off after 10 mins. This was followed by 3 hr incubation (temperature was 37 ± 3 °C) with 20 μL of 0.5 mg/ml primary antibody in PBS buffer (pH = 7.0) and 0.05% Tween-20. The electrodes were then washed with 0.05% Tween-20 for 1 min and then 1 min with pH 7.0 PBS buffer. Each electrode was then incubated with 20 μL of 2% BSA in 0.05% Tween-20 and PBS buffer for 1 hr at 37 ± 3°C . The electrodes were then washed with 0.05% Tween-20 for 1 min each and then 1 min each with PBS buffer (pH = 7.0), followed by incubation with a 10 μL drop of pH 7.0 PBS buffer containing various concentrations of PSA and serum containing various concentrations of PSA for1 hr followed by washing with 0.05% Tween-20 in PBS and PBS solution as described above. The next step was incubated again with 10 μL drop of Ab2 Tag. SEM preparation begins with samples of bare MWCNTs and Ab2 tags diluted 10 times using deionized water. Gold sputter coated silicon wafers were prepared by adhering them with silver paint to a metallic conductive mounting platform. 10, 5, and 2 Pl of each sample were placed on top of the gold sputter coated silicon
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wafers and dried at room temperature. Samples were then placed into a vacuum environment for a day before characterization using SEM.
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A CHI 660 electrochemical workstation was used for cyclic voltammetry and amperometry at ambient temperatures (22 ± 2 °C) in a three-electrode electrochemical cell. Immunosensor was placed in the electrochemical cell containing 10 mL (pH 7.0) PBS buffer and 1 mM hydroquinone. Rotating disk amperometry at 3000 rpm was done at -0.3 vs. SCE with 40 ȝ0 H 2 O 2 added to detect the HRP labels, as these conditions gave optimum sensitivity.
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Scanning Electron Microscopy A Zeiss DSM982 Gemini Field Emission Scanning Electron Microscope (FESEM) with a Schottky electron source was used. The Gun Chamber was Ion pumped while the specimen chamber was evacuated with a turbo molecular oil free pump backed by a rotary vane pump. M i c r o s c o p y wa s c o n d u c t e d a t a m b i e n t temperature (22 ± 2 oC) in an evacuated chamber at pressures < 9X10-9hPa. Ion beam focusing was set at 3 mm with an ion power source of 2.00kV.
3.0 Results and discussion 3.1 Electrochemical The schematic diagram of the above discussed electrochemical immunoassay is shown in Fig 1.
Fig 1 Schematic Diagram of Amplified Electrochemical Immunoassay Amperometric data was taken on assays containing PSA in buffer solution (Fig. 1) and PSA in serum (Fig. 2).
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Figure 2. Detection of PSA in Serum.
Fig. 1 signals are produced with an injection of H2O2 with final concentration of 40ȝ0 and hydroquinone (HQ) with final concentration of 1 mM into 10ml of PBS solution (pH=7.0). Electrodes were rotated using a Rotating disk at 3000RPM. As can be observed from Fig 1 and 2, as concentration of PSA increases from 0.004 ng/mL to 0.1 ng/mL, amperometric signals increase proportionally due to the increase in binding of HRP-Ab2-MWCNTs with PSA immobilized on the electrode surface. Non Specific Binding (NSB) has been minimized based on previous work [12] to ensure the majority of the signal comes from the PSA bound to electrode and Ab2 Tag. Controls of a full assay built on PG without SWNT forest at 100 pg/mL PSA showed a 2~3 times smaller signal than assay built on SWNT forest, clearly indicating the advantage in using SWNT forest to facilitate vectorial electron transfer between enzyme and electrodes.
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Fig. 2 also demonstrates that our method can work beautifully in a more complex matrix such as serum, suggesting the promising future application of this method in point-of-care diagnostics.
better conduction of electrons from the FESEM electron source. This conduction builds up less charge on bare MWCNT surfaces compared to protein-MWCNT conjugate due to materials bound to MWCNTs such as HRP and Ab2.
3.2 Characterization Using SEM
4.0 Conclusions
Fig. 3 and fig. 4 are typical SEM images of different MWCNT’s before and after they are conjugated with HRP and Ab2.
Amplified electrochemical immunoassay shows highly sensitive and reproducible detection of down to 0.004 ng/mL of PSA, opening an promising method which may lead into the detection of protein cancer biomarkers not only in serum but in cellular tissues. Detection of PSA down at the cellular level can prevent or isolate growth of cancerous tissue with non-invasive treatments making cancer such as pro state cancer less life threatening.
Figure 3. SEM image of a typical bare MWCNT. This bare MWNT was from a 5 Pl sample of bare MWCNT’s placed onto a gold sputtered silicon wafer and dried.
Future work will include detection of PSA in tissue and serum sample of patients, full characterization of Ab2 tag to ensure reproducibility of this method, development of other biosensors containing different biomarkers, and adopting these multi biosensors to an array based format. Acknowledgements This work was supported by US Army Research Office (ARO) via grant DAAD-02-1-0381. The authors thanks Mr. James Ramanow at UConn Department of Physiology and Neurobiology for assistance with SEM measurements. References
Figure 4. SEM image of a typical MWCNT containing Ab2’s and HRP’s attached . This bare MWNT was from a 5 Pl sample of bare MWCNT’s placed onto a gold .sputtered silicon wafer and dried
Several samples of Ab2 tag and bare MWCNT’s were observed using FESEM, reproductive results are illustrated in Figures 3 and 4. As can be observed from the figures, MWCNTs conjugated with proteins shows an average of 8 nm increases in diameter comparing to bare MWCNTs, suggesting the existence of a thin film of protein. Also noteworthy is the better contrast of bare MWCNT comparing to protein covered MWCNT at the same magnification scale (fig. 4), which could be reasonably attributed to the fact that bare MWCNTs have
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cancer. Int. J. Gynecolog. Cancer, 2003, 13 (Suppl. 2) 133-139. [8] Wilson, D. S.; Nock, S. Recent developments in protein microarray technology. Angew. Chem. Int. Ed. 2003, 42, 494-500 and references therein. [9] Wagner, P. D.; Verma, M.; Srivastava, S. Challenges for biomarkers in cancer detection. Ann. N. Y. Acad. Sci. 2004, 1022, 9-16. [10] Chattopadhyay, D.; Galeska, I.; Papadimitrakopoulos, F. Metal assisted organization of shortened carbon nanotubes in monolayer and multilayer forest assemblies. J. Am. Chem. Soc. 2001, 123, 9451. [11] Yu, X.; Kim, S. N.; Papadimitrakopoulos, F.; Rusling, J. F. Protein Immunosensors Using SingleWall Carbon Nanotube Forests with Electrochemical Detection of Enzyme Labels, Molecular Biosystems, 2005, 1, 70-78
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