Functionalised Silicon Microchannel Immunosensor

Functionalised Silicon Microchannel Immunosensor With Portable Electronic Readout for Bacteria Detection in Blood C.RoyChaudhuri*, R.Dev Das,S.Dey

S.Das

Department of Electronics and Telecomm. Engineering Bengal Engineering and Science University, Shibpur Howrah, India [email protected]

Department of Neurobiology Indian Institute of Chemical Biology, Jadavpur Kolkata, India [email protected]

Abstract—This paper reports the development of functionalized silicon microchannel immunosensor with optimized electrode geometry and portable electronic interface towards specific detection of E.coli in blood samples for the first time. The sensor with simple electrode geometry fabricated by low cost screen printing method detects 103CFU/ml E.coli in spiked blood samples in 30 minutes without any labeling and with less than 3% false positive results which is of great clinical significance. To avoid biofouling, an initial centrifugation is done for the first 15 minutes of the total 30 mins which helps in improving specificity. The battery operated portable electronic circuit is designed with zero offset features to take into account the variability in the sensor fabrication for faithful display of bacteria concentration range. Hence, in comparison to the existing reports, the proposed immunosensor system has an optimum combination of the essential features required for a commercial biosensor.

I.

INTRODUCTION

The development of a biosensor for rapid detection of pathogenic bacteria in blood is of great importance for the physicians. The desirable features of a commercial biosensor for this application include clinically significant sensitivity of 103CFU/ml, specificity of detection, rapidity, less operator dependence, portability and low cost[1,2]. Of the available commercial instruments for the rapid detection of bacteria, Unilite, UK has a detection limit of 103CFU/ml but it lacks specificity and portability [2]. Most of the reported optical biosensors like fluorescence, SPR and others are non-portable and expensive and nitrocellulose strip based colorimetric method is less sensitive[3,4,5]. The cost intensive electrical biosensors fabricated by photolithographic patterns which have been tested with blood suffer from the problem of biofouling leading to reduced specificity and reproducibility [6]. To avoid this, labels like photocleavable crosslinkers and microfluidic channel based purification are used but at the cost of improved complexity and expenses. Recently label free

functionalized silicon microchannels of macroporous silicon formed by electrochemical etching have been found to detect 103CFU/ml bacteria with good specificity and improved sensitivity in pure culture with simple electrode geometry fabricated by screen printing method [7, 8]. But these silicon microchannels have been tested with blood sample before. This interesting feature has motivated the authors to explore this platform with blood samples. In this paper non-infected blood samples have been taken and spiked with different concentration of E.coli. A simple centrifugation procedure has been followed to separate and preconcentrate the bacterial cells. The sensor platform has been interfaced with portable electronics readout for direct display of the bacterial concentration range. II.

MATERIALS AND METHODS

A. Preparation of Silicon Microchannle Based Platform For fabrication of silicon microchannels, p-type silicon wafers of 10-20 Ωcm resistivity are cleaned by standard procedure. The cleaned silicon wafer is then etched anodically in a double pond electrochemical bath. The etching is carried out under constant current source of current density 2mA/cm2 with an electrolyte mixture of HF (48 wt%) in DMSO in the ratio of 1:9. The time of etching is kept at 60mins to obtain porous layer thickness of around 8 µm as reported in [7]. The porosity of the porous silicon is obtained to be 55% for all the samples. These microchannels are next oxidized for silanization and antibody immobilization. For uniform thermal oxidation of macroporous silicon with 0.9 µm oxide growth, a dry–wet–dry sequence has been followed in an oxidation furnace for 3 hrs at 1000oC. By this method, the silicon crystallites are partially oxidized which also maintains sufficient opening of the pores after oxidation. Electrode fabrication has been carried out using screen printing method by aluminium ink (FERRO 53120) on

oxidized silicon microchannels. The aluminium metal contacts are next cured at 450oC for 45 s in nitrogen ambient. After that, gold metal is evaporated on Al metal contacts using copper stencil mask for attaching bond wires (Kulicke and Soffa Model no. 4523A0) for external contacts. The picture of the sensor with fabricated electrode is shown in Fig.1.

Fig.1 Picture of the sensor chip

B. Antibody Immobilization Procedure After oxidation, the oxidized macroporous silicon samples are treated with mercaptopropyltrimethoxysilane (MTS) silane. The optimized parameters reported in [9] have been applied here for monolayer silane film coating. 4Maleimidobutyric acid N-succinimidyl ester (GMBS) has been used as a hetero bifunctional reagent for attachment of cross linker. The succinimidyl group of GMBS reacts with the -SH group of silane. The GMBS coated samples are dipped in 0.1mg/ml concentration of E-coli O157 antibody solution in 20mM phosphate buffer saline (PBS) and then incubated at room temperature for 30 minutes to allow complete binding for antibody immobilization. The nonspecifically adsorbed antibody is removed by washing in PBS. Further details of the process have been reported in [8]. C. Separation of bacterial cells from blood sample 6 ml blood sample is taken in a 15 ml centrifuge tube and then spiked with E.coli bacteria into the blood. The final concentration of bacteria into the blood is 103 CFU/ml. After that, the E.coli bacteria contaminated blood is centrifuged at 1000 RPM for five minutes to precipitate the blood cells but not the bacteria cells. Then 3 ml of supernatant plasma containing bacterial cells is collected and centrifuged at 5000 RPM to precipitate the bacterial cells. After that, 2.7 ml supernatant plasma is removed and 3 ml bacteria containing plasma is collected. Then desired amount of PB solution is added so that the final concentration of bacterial cells in to is 5×104 CFU/ml. For other concentration of bacteria, the same steps are followed for detection of unknown bacterial infection load in blood. The authors would like to acknowledge Department of Science and Technology, Instrument Development Programme, Government of India for funding this research.

D. Specificity Testing For specificity testing, assays of, E-coli and S.Typhimurium cultures have been prepared with serial dilutions. Four types of tests have been performed for specificity testing. Test I is a test in pure culture where the sensor is tested for the target antigen E-coli only.Test II is a test in pure culture of S.Typhimurium. Test III is a mixture of E.coli and S.Typhimurium culture experiment. Assays on dilution series of target and non-target bacteria in the same sample are conducted to assess the specificity of the biosensor in mixed cultures. E. Impedance Measurement All the measurements have been carried out in triplicate to estimate the reproducibility of the silicon microchannel sensors. The sensors are initially characterized electrically with Agilent 4284A impedance analyzer between 100Hz and 1MHz to estimate the sensitivity of the sensors with different E-coli concentration. An applied voltage of 50mV has been chosen for measurements to avoid any random fluctuation of resistance and capacitance at the electrode–electrolyte interface. All the sensors are exposed to blank PBS as the control solution with a micropipette and the steady state readings are recorded by the data acquisition card interface with PC available with Agilent 4284A. Simultaneously, the output of the signal conditioning circuit displayed in a LCD is recorded corresponding to the frequency at which the sensitivity has been found to be maximum. The sensors are then exposed to the bacteria culture medium with different concentration and are incubated at 37◦C for 10 min for binding with the antibody. The remaining bacterial solution is then washed off. The impedance readings are next recorded again by a LCR meter and the final output of the signal conditioning circuit showing the percentage change in impedance is observed. The total flowchart for measurement is shown in Fig.2

Fig.2 Brief flowchart for operation of the system

III.

RESULTS AND DISCUSSIONS

A. Measurement using standard impedance analyzer The impedance of the silicon microchannel sensor for an initial bacterial concentration of 103CFU/ml is shown in

Fig.3a with frequency. The percentage change in impedance for different bacterial concentration in blood is shown in Fig.3b with frequency. It is observed from Fig.3a that the impedance decreases significantly with frequency both before and after bacteria capture. This is primarily because the overall impedance comprises of the double layer impedances at the electrode-electrolyte and the SiO2-electrolyte interface. Further, the double layer impedances at the electrodeelectrolyte and the SiO2-electrolyte interfaces decrease with frequency due to the surface roughness present on the electrode and the SiO2 surfaces. At low frequency, the ions in the solution get enough time to cover the crests and troughs of the rough surface significantly compared to the high frequency operation [9]. 2.2

Before Bacteria attachment After Bacteria attachment

Impedance in K Ohm

2.0 1.8

The sensitivity of the silicon mirochannels at different frequency is obtained by computing the fractional decrease in impedance after bacteria capture from Fig.3a. The fractional decrease in impedance at 100Hz is around 15%, at 500Hz is 14.8%, at 1kHz is 14%, at 10kHz is 13% and so on for input concentration of 103CFU/ml as observed from Fig.3b. Similarly the maximum sensitivity at 104CFU/ml and 105CFU/ml at 100Hz is observed to be 50% and 95% respectively. This is primarily because with increase in frequency, the double layer impedances both at the electrodeelectrolyte and SiO2-electrolyte interfaces decrease as power law and the solution resistance becomes more significant after 1kHz. However the solution resistance has a tendency to increase after bacteria capture since the bacteria molecules are highly resistive [10]. This reduces the fractional decrease in the overall impedance with increasing frequency. B. Measurement with Signal Conditioning Units The block diagram of the signal conditioning unit is shown in Fig.4. DC offset and amp reduction

Buffer

1.6 sine wave generator

1.4 1.2

battery

1.0 100

1000

10000 Vo

Frequency in Hz

sensor

Fig. 3a Impedance with frequency before and after bacteria attachment for an input concentration of 103CFU/ml. Microcontroller with built in ADC

100

in impedance % decrease

LCD display

Fig.4 Block Diagram of the signal conditioning circuit

90 80

4

10 CFU/ml 5 10 CFU/ml 3 10 CFU/ml

70 60 50 40 30 20 10 100

Buffer

1000

10000

100000

1000000

Frequency(Hz) Fig.3b % decrease in sensitivity with frequency for different bacteria concentration

The sine wave generator chip is used to generate a sinusoidal wave of variable frequency but its output has a dc offset voltage. To eliminate this dc offset voltage, two capacitors are connected in parallel and two buffers are used to increase the input impedance. Two transistors are used for frequency setting and which is controlled by microcontroller. The amplitude of the sine wave is reduced by using a voltage divider circuit. An inverting amplifier is used after the buffer part to get corresponding output voltage in accordance with the sensor resistance. The input voltage is kept within the range of 50 mV. In the circuit, sensor is used as an input resistance of the inverting amplifier and feedback resistance of this amplifier is fixed at 10kΩ. The output voltage will change in accordance with the sensor resistance and gets amplified almost 10 times since the sensor resistance is of the order of 1kΩ. Assuming sensor resistance is varying in between 1kΩ to 10kΩ and the output voltage will vary in the

range 1000 mV to 50 mV. Finally the output voltage is fed to the ADC port of the microcontroller and percentage change is computed. The percentage change in impedance along with the bacterial concentration is displayed as shown in Table 1.The picture of the signal conditioning circuit is shown in Fig.5.

It is observed from Table1 that the readings of the circuit match closely that obtained from a precision LCR meter. Further the sensor system is also found to be specific, stable and reproducible which are important requirements for commercialization. ACKNOWLEDGMENT

Bacterial strains in blood

Only E.coli in 103CFU/ml Only S.Typhimurium in 103CFU/ml 1:1 mixture of E.Coli and S.Typimurium in 103CFU/ml Only E.coli in 104CFU/ml

Sensitivity from LCR meter at 100Hz 15%

Sensitivity from circuit

Range display (CFU/ml)

14.5%

>1000

4.8%

4.7%

1000

The author would like to acknowledge Prof.H.Saha of Center of Excellence for Green Energy and Sensor Systems of Bengal Engineering and Science university, Shibpur for his valuable advise. The authors are also grateful to Mr.Amit Chanda of Amateur World for his help in the development of the PCB board. REFERENCES [1]

50%

48%

>10000

Table 1 Sensitivity readings of LCR meter and circuit.

Fig. 5 Picture of the signal conditioning circuit

M.S. Mannoor, S.Zhang, A.James Link, M.C. McAlpine, “Electrical detection of pathogenic bacteria via immobilized antimicrobial peptides”, PNAS, vol. 107, no. 45, pp.19207-19212, 2010. [2] D.Ivnitski, I.A.Hamid, P.Atanasov, E.Wilkins, “Biosensors for detection of pathogenic bacteria”, Biosensors and Bioelectronics, vol. 14, pp.599-624, 1999. [3] Yuval Ebenstein & Laurent A. Bentolila, “Single Molecule Detection: Focusing n the Objective”, Nature Nanotechnology,vol.5, pp.99-100, 2010 [4] Joachim Jose, Ji-Won Chung, Byoung-Jin Jeon, Ruth Maria Maas, Chang-Hoon Nam, Jae-Chul PyunHhj, “Escherichia coli with autodisplayed Z-domain of protein A for signal amplification of SPR biosensor”, Biosensors and Bioelectronics, vol 24, issue 5, pp.13241329, 2009. [5] Primchanien Moongkarndi, Ekkarat Rodpai, Sasitorn Kanarat, “Evaluation of an immunochromatographic assay for rapid detection of Salmonella enterica serovars Typhimurium and Enteritidis”, Journal of Veterinary Diagnostic Investigation, vol. 23 no. 4,pp.797-801, 2011. [6] E.Stern, A.Vacic, N.K.Rajan, J.M.Criscione, J.Park, B.R.Ilic, D.J.Mooney, M.A.Reed,T.M.Fahmy, “ Label Free Biomarker Detection in Whole Blood”,Nature Nanotechnology,vol.5, pp.138-142, 2010. [7] R. Dev Das, C.RoyChaudhuri, S.Maji, S.Das, H.Saha, “Macroporous silicon based simple and efficient trapping platform for electrical detection of Salmonella typhimurium pathogens”,Biosensors and Bioelectronics, vol.24,pp.3215-3222, 2009 [8] R.DevDas, A.Dey, S.Das, C.RoyChaudhuri, “Interdigitated Electrodeless High Performance Macroporous Silicon Structure as Impedance Biosensor for Bacteria Detection”, IEEE Sensors, vol.11,pp.1242-1252, 2011. [9] R. Dev Das, S.Maji, S.Das, C.RoyChaudhuri,, “Optimization of covalent antibody immobilization on macroporous silicon solid supports”, Applied Surface Science, vol.256, pp.5867-5875, 2010 [10] Y.B. Wang, R.K.Yuan, M.Willander, “Capacitance of semiconductor electrolyte junction and its frequency dependence”, Appl. Phys. A, vol.63, pp.481-486, 1996.