Rapid Detection of E.Coli Bacteria using Potassium-Sensitive FETs in CMOS

Nasim Nikkhoo and P. Glenn GulakDept. of Electrical and Computer Eng., Univ. of Toronto

Toronto, Canadanikkhoo@eecg.toronto.edu, gulak@eecg.toronto.edu

Karen MaxwellDept. of Medical Genetics and Microbiology, Univ. of Toronto

Toronto, Canada

Abstract—An integrated bacteria detection chip is implemented in 0.18µm CMOS technology. The chip has been tested using phages as biological detecting elements along with CMOS ion-selective FETs to detect the presence of a specific strain of E.coli. The chip successfully identifies the presence of living bacterial strains sensitive to the T6 phage in less than 30 minutes. Potassium-sensitive ISFETS are implemented using CMOS ISFETs and a post-processed potassium-sensitive PVC-based membrane on the top layer.

I. INTRODUCTION

There is increasing demand for systems that detect bacteria with good selectivity and sensitivity, in applications ranging from medical diagnosis to water and food inspection. Traditional culturing methods take several hours to days to give results. They require trained personnel to perform the tests as well as expensive laboratory equipment.. Techniques using DNA amplification and detection like polymerase chain reaction (PCR) can provide results in several hours. DNA-based detection approaches offer the potential for miniaturization and have been integrated on silicon chips. The disadvantages of these methods are laborious sample preparation and more importantly, their inability to distinguish between living and dead bacterial cells. The need for an integrated low-cost system that can selectively detect living bacterial organisms still remains.

We present a bacteria identification sensor integrated in CMOS using phages as biological sensing elements combined with ion-selective FETs (ISFET) implemented in conventional CMOS technology to accurately detect specific strains of living bacteria in less than 30 minutes. This work can provide a simple low-cost platform for bacteria detection using sensitive low-noise electronic circuits combined with bacteriophages as biological sensing elements with the specificity required to sense different strains of living bacterial cells accurately.

Bacteriophages (phages) are types of viruses that infect bacteria. They are ubiquitous in nature and are very specific to their targets. Phage libraries with specificities for a wide variety of bacteria are available commercially. Phages are inexpensive and can be made at high concentrations (to around 1012 pfu/ml) and stored for several years. In addition they have no special storage and handling requirements.

Ion-selective FETs (ISFET) have been first described in [1]. They are low-cost integrated alternatives for ion-selective electrodes (ISE) and have been extensively used and reviewed in several papers [4]. Conventional ISFETs are fabricated very similar to regular MOSFETs except that no gate metal is deposited and an ion-sensitive membrane is deposited as the gate dielectric. The gate connection is

separated from the chip by the aqueous sample and a reference electrode is inserted in the sample to define the sample potential. Most ISFETs are used as pH sensors because of the hydrogen sensitivity of SiO2 used in MOS processes as gate oxide. In MOSFETs, the threshold voltage of the transistors is completely defined by the process parameters. ISFETs use the same process technology as MOSFETs and hence have the same constant part of the threshold voltage. In addition to MOSFET Vt , an ISFET threshold voltage has a constant potential of the reference electrode, Eref, and the interfacial potential at the solution/oxide interface [4]. The interfacial potential consists of a constant surface dipole potential plus a pH-dependent term that changes with the concentration of H+ ions as described in (1) where denotes the pH dependent surface potential and � is the constant dipole potential. The rest of the parameters are standard CMOS process parameters also defined in [4]. The variations of with respect to pH are illustrated in (2). Depending on the quality of the oxide and its surface buffer

capacity, ↵ can vary from 0 to 1. With the value 1, the ISFET has full Nerstian sensitivity of 58.2 mV/dec at 20℃.

In [2] pH-sensitive ISFETs were extended to detect potassium ions by applying a valinomycin/plasticizer/polyvinylchloride membrane on top of the gate. This was a significant step forward in using ISFETs as multiple ion sensors. CMOS-compatible ISFETs were introduced, as in [3], that have stacks of metals and vias connecting the gate in CMOS transistor to the top metal. The passivation layer on the top metal is the SiO2/Si3N4 mixture that can directly be used as pH-sensitive membrane. CMOS ISFETs can be viewed as the series combination of the sensitive membrane and the conventional MOSFET transistor. Because of their low-cost they are extensively used. CMOS-based ISFETS are also utilized in this work.

II. SYSTEM DESIGN

The implemented bacterial sensor system is comprised of four building blocks as detailed in Fig. 1. The biological transducer generates the detectable signal in the electrical transducer. It consists of the test sample plus phages specific to our desired bacterial target. The output signals from this block are the released potassium ions in the sample that need

� = �2.3↵RTF �pH (2)

V

t

= E

ref

� + �

sol � �

q

� Q

ox

+Q

ss

+Q

B

C

o

x

+ 2�f

(1)

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to be converted into an electrical signal using the electrical transducer (ISFET) block. The analog circuitry that reads the signal from the electrical transducer is the analog front-end whose output is then converted to a digital representation. The

digital output is transferred to a PC and is further processed and visualized. These blocks are further described in the following sections.A. Biological Sensing System

Detection of a specific strain of bacterial cells is achieved using the bacteriophages that infect that strain. The interaction between the phage tail fibers and receptors on the bacterial cell surface results in attachment of the phage to its target. After its attachment, the cell membrane goes through a

depolarization phase and becomes permeable to specific ions especially potassium ions [6]. Phage DNA is transferred from the head part of the phage to inside the cell. Following this phase there is an efflux of ions from inside the cell to the medium outside the cell as shown in Fig. 2. Potassium ions are the dominant ions inside the cell that flow to the outside sample as a result of phage infection; hence this transient efflux of K+ is used as the signal to be detected in an electrical transducer using potassium sensitive ISFETs.

B. Transducer DesignThe outputs of the biological transducer are the potassium

ions. The level of potassium build-up after the addition of the phages to the sample is proportional to the concentration of the present infected bacterial cells. CMOS-based ISFETs are the ideal transducers for sensing a specific ion in the sample.

They offer rapid response time, small size and can be integrated along with other required readout and signal processing circuitry on the same chip; hence low noise and small form-factor systems can be implemented with multiple measurement channels. Fig. 3 shows the block diagram of the implemented potassium-sensitive system on the chip. Conventional PMOS transistors are integrated and their gates are connected through vias and multiple levels of metal to the top metal layer with the sizes shown in Fig. 3. The passivation layer on top of the top metal is a mixture of SiO2 and Si3N4

and is only suitable for pH sensors. In order to achieve the desired potassium sensitivity, a PVC-based potassium sensitive layer based on the composition identified in [3] has been prepared and deposited on top of the chip.

The potassium-sensitive membrane utilizes a macrocyclic substance from a group of depsipeptides, valinomycin, as the ion-exchanger. This antibiotic forms complexes with alkali metal ions and is highly selective to potassium K+ ions. It is a member of group of natural neutral ionophores. Fig. 4 shows the structure of the valinomycin, where the potassium ion is held inside the cylinder that fits the size of K+ ion. The ion-exchanger is dissolved in an organic solvent like tetrahydrofuran (THF) along with polyvinylchloride (PVC) and plastizer dioctylphthalate that together build a porous membrane to hold the valinoymicin inside the membrane structure.

In order to prepare the K+ sensitive ISFET, the chips containing the ISFET transistors were first encapsulated using biologically-inert epoxy. All bond-wires, bonding pads, and chip scribe lanes were covered leaving only the electrodes

Figure 1. Left: System Functional Block Diagram. Right: Detailed System Diagram.

Figure 2. Left: Typical Phage Structure. Right: Infection of the sensitive bacterial cell by the phage, DNA injection and efflux of K+ ions. (Not drawn to scale.)

Figure 3. ISFET cross-section implemented in CMOS with the potassium sensitive membrane on the top surface.

Figure 4. Valinomycin structure with K+ ion.

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exposed. The solution of the membrane components in THF solvent was prepared under a fumehood and mixed thoroughly.

In order to enhance the attachment of the membrane to the chip surface, surface cleaning, drying and silanization was performed as follows. The encapsulated chip was cleaned using acetone and isopropanol and then deionized water. It was dried using nitrogen gas and placed in an oven at 110℃ to dry. After further drying at room temperature a small droplet of P20 was deposited on the surface of the chip as an adhesion promoter under the fumehood. After drying of the promoter, droplets of the membrane solution were dispensed on top of the electrodes 2 to 3 times with 5-minute intervals in between applications to create a smooth membrane layer with a thickness in the range of 100 to 300µm without any pinholes. The membrane was left to dry (at room temperature) for several hours. This process prepared the required K+ sensitive ISFET as the electrical transducer.

C. Analog Front-End Circuitry and Measrement BlockFig. 5 shows the drain-source follower circuit used to read

the ISFET threshold variations. Both Vds and drain current of the ISFET are fixed to 0.5 volts and 100µA, respectively. A fixed DC voltage is applied to the solution being in contact with the ISFET using a reference electrode. The source voltage of the ISFET is measured using the readout circuit.

The gate source Vgs of the PMOS is defined by the process parameters of the transistor when fixed Vds and ID are applied. The changes in the threshold voltage of the ISFET, because of K+ concentration variations, are reflected in the source voltage. As the concentration of K+ increases, Vs also increases. For testing purposes both current ID and Vds of the ISFET are var iable to enable complete ID-VDS characterization curves.

The prototype ISFETs were implemented in 0.18µm CMOS technology and the readout circuits were implemented outside the chip on the test PCB. The chip die photo is shown in Fig. 6. This chip contains several measurement channels with different sized electrodes as well as other test circuitry. Outputs of the ISFET readout circuits were converted to a digital representation and transferred to a PC using a NI data acquisition board and visualized real-time using Labview.

Because of the compatibility of ISFETS with standard CMOS technology, readout circuitry can be easily integrated on the chip, as in [2]. Ultra-low-power ADCs with low sampling rates have been implemented [5] and can be integrated on-chip creating a low-cost, single-chip integrated realization.

III. EXPERIMENTAL PROTOCOL AND SETUP

Live bacterial cells and phages were used on the chip in the experiments. Before starting the experiments, the chips were cleaned and fresh potassium-sensitive ISFET chips were prepared as explained in the previous section.

The measurements were done using two strains of E.coli K12 bacterial cells. The positive control experiments were performed using E.coli K12 BL21 (DE3-∆tail), called BL21 here for simplicity, which are sensitive to our phage. The negative control experiments were performed using bacterial cells E.coli K12 BW25113 with TSX- , called TSX- here, which are insensitive to the phage. The phages used throughout the experiments were T6 phages.

Bacterial cultures were prepared overnight using frozen cultures inoculated in Luria broth (LB) growth medium. The overnight cultures were then inoculated to make fresh samples for the day of the experiments. The growth of the cells was monitored using a spectrophotometer that read the optical density (OD) of the samples compared to a blank LB sample. For both experiments, cultures were used as their OD reached 1.2. Using previous experiments, we have shown that this OD translates to a cell concentration of approximately 109 cfu/ml. After their growth, bacteria were washed and resuspended twice in a clean medium without potassium where they would remain alive without replication.

The phages were prepared beforehand and stored in the 4℃ refrigerator for several months without any drop in their concentration. The concentration of phages used in the experiments was 8×1010 pfu/ml.

Figure 5. Left: ISFET readout circuit. Right: Circuit used to vary the ISFET transistor drain current.

Figure 6. Chip micrograph.

Figure 7. Experimental setup.

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The measurements were performed inside a dark chamber since ISFETs are sensitive to light. The cells as well as the phages were transferred to the top surface of the chip via tubes connected to their respective syringes outside the dark chamber. The syringes were manipulated manually for transfer and mixing of the cells and phages. Fig. 7 shows the block diagram of the experimental setup.

IV. EXPERIMENTAL RESULTS

Two sets of experiments were performed using BL21 and TSX- cells, respectively. A positive control experiment was performed by mixing the BL21 cells with T6 phages. BL21 cells are sensitive to T6 phage and we expect infection and release of potassium after mixture. A negative control experiment was performed by mixing the TSX- cells with T6 phages. TSX- cells are insensitive to T6 phages; hence no infection and no potassium release is expected. At the beginning of each experiment, 0.5ml of the bacterial cells (BL21 for positive control and TSX- for negative control) were transferred to the ISFET chip surface and the measurement started. ISFET outputs were recorded for 5 minutes without addition of the phages. This step can help identify the ISFET drift and obtain a DC baseline in the measurements. After 5 minutes, 20µL of T6 phages were added to the sample and mixed thoroughly for 1 minute. The mixture was left on the chip for 30 minutes. Data was continuously recorded from the start of the measurement, through the mixing phase and also after mixing until the end of the experiment. The results shown in Fig. 8 were processed in Matlab with a 0.3 Hz low-pass filter. The results show ∆V changes to remove any DC baseline differences between the

experiments. ISFETs are known to have inferior drift and hysteresis problems compared to ISE counterparts. In this detection scheme, since the potassium level changes are measured before and after the addition of the phages, drift or hysteresis and threshold voltage variations between ISFETs do not interfere with results and can be disregarded or post processed from the measurement data. Here we have removed the DC baseline, but drift is present and shown in Fig. 8.

Fig. 8 shows the results of both positive and negative control experiments. As can be observed, the output increases

as a result of the efflux of potassium ions from the live infected cells in the positive control test. In the negative control experiment, the output continues to have the downward ISFET drift similar to that present before the addition of the phages.

The results show that the integrated potassium-sensitive chip can successfully detect the presence of a specific E.coli strain BL21 using the specific T6 phage. The designed sensor system can tolerate the non-idealities present in ISFET integrated circuits like drift, hysteresis and threshold variations without affecting the results. Simple signal processing circuitry can be added to enhance the visualization. Post-processing circuitry can readily be integrated on-chip to indicate the detection of the target bacteria (present / not-present) in less than 30 minutes. Inspection of the measured results confirm that the detector outcome can accurately be determined within the first 10 minutes after mixing the bacteria and phage.

V. CONCLUSION

A 0.18µm CMOS integrated ISFET chip provides an integrated lab-on-chip solution for the rapid identification of live bacteria. It combines the specificity of phages as the biological detecting elements and the sensitivity afforded by ISFET and CMOS circuitry implemented in conventional CMOS technology whose electrodes are post-processed with a potassium-sensitive membrane. The implemented system along with measurement results show that the system can be exploited to achieve a low-cost, portable bacterial identification system. Non-idealities such as drift in ISFETs have no material effect on the integrity of collected data affording a detection time of less than 30 minutes.

ACKNOWLEDGEMENTS

Financial support by CIHR, NSERC and fabrication support by CMC is gratefully acknowledged.

REFERENCES

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[3] J. Bausells, J. Carrabina, A. Errachid, A. Merlos, “Ion-sensitive field effect transistors fabricated in a commercial CMOS technology”, Sens. Actuators B, Vol. 57, 1999, pp. 56–62.

[4] P. Bergveld, “Thirty years of ISFETOLOGY: What Happened in the Past 30 Years and What May Happen in the next 30 years,” Sensors and Actuators B, Vol. 88, 2003, pp. 1–20.

[5] M. D. Scott, B. E. Boser, and K. S. J. Pister, “An Ultralow-Energy ADC for Smart Dust,” IEEE J. Solid-State Circuits, Vol. 38, no. 7, pp. 1123–1129, Jul. 2003.

[6] P. Boulanger and L. Letellier, “Characterization of Ion Channels involved in the Penetration of Phage T4 DNA into Escherichia Coli Cells,” J. of Biological Chemistry, Vol. 263, No. 20, 1988, pp. 9767-9775.

Figure 8. Output Voltage of the ISFET for the positive and negative control experiments.

Phage Added

Mixing

Cells Alone Cell Mixture with Phage

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