WO2014070110A1 - Device and method for detecting an intestinal parasitic protozoan - Google Patents

Abstract

This invention relates to the detection of intestinal parasitic protozoan. In particular, the invention relates to a device and method for detecting Cryptosporidium parvum oocysts in a sample. The device comprising: (a) a carbon substrate, wherein an antibody or an antibody fragment specific to an epitope of the parasite is immobilized on the substrate; and (b) an electrical circuit configured to monitor conductance across the substrate, wherein the presence or absence of the parasite is detected based on a change in the conductance. The method comprising: (a) contacting the sample with an antibody or an antibody fragment that binds specifically to an epitope of the parasite; and (b) detecting the presence of the parasite-bound antibody in the sample.

Description

DEVICE AND METHOD FOR DETECTING

AN INTESTINAL PARASITIC PROTOZOAN Field of the invention

This invention relates to the detection of intestinal parasitic protozoan. In particular, the invention relates to a device and method for detecting Cryptosporidium parvum oocysts in a sample.

Background of the invention

C. parvum is a common intestinal parasitic protozoan that causes gastroenteritis in man and animals. It poses high risks to drinking water supply because of its ubiquitous distribution in water where it can remain viable for infection even after 6-8 months and their oocysts are resistant to harsh environment conditions. C. parvum infects its host by invading the intestinal and urogenital systems. C. parvum organisms may be transmitted in a variety of ways including via contaminated food or water, animal to animal contact, via farm animals such as sheep and calves, or alternatively by oocysts in feces. Human infections generally result from zoonotic spread, person-to-person contact, fecal-oral contact, oral-anal contact or waterborne transmission.

Typically, C. parvum organisms are small (2 to 6 μπι) spherules that inhabit the microvillus border of the intestinal epithelium arranged in rows along the brush border of the jejunum. After introduction into the intestine, C. parvum sporozoites attach to the microvilli surfaces and reproduce by schizogony (asexually). The resulting infective oocysts are passed into the intestinal lumen and passed in the feces. Following ingestion of the oocysts by another vertebrate, the oocysts release sporozoites that attach themselves to the epithelial surface and initiate a new cycle of infection. As C. parvum organisms invade the surface of intestinal cells, the host experiences symptoms such as reduced appetite, severe diarrhea and chronic fluid loss. In normal hosts, the onset of the disease is explosive, with profuse, watery diarrhea and abdominal cramping that lasts from 4 to 14 days following exposure. The symptoms generally persist for 5 to 11 days, and then rapidly abate as remission of the parasite occurs in about 10-15 days. There is no effective and specific therapy available at present. Although some patients have responded positively to therapy with conventional antibiotics such as spiramycin and paromomycin, the result of infection is frequently fatal for immuno-compromised individuals. In fact, cryptosporidiosis is one of the predominant causes of death in immuno-compromised patients.

In light of the potential disastrous consequences of C. parvum infection, sensitive, efficient methods for detecting C. parvum contamination are necessary. In humans, the typical source of cryptosporidiosis is contaminated water, therefore safeguarding water supplies is a primary goal.

Standard procedures such as EPA method 1622 and 1623 are typically used to process contaminated water. Conventionally, C. parvum oocysts (Cp. oocysts) can be detected by various detection techniques such as acid-fast staining, immunofluorescent (IF) antibody staining, flow cytometry and polymerase chain reaction (PCR).

Each detection technique has different features and the most frequently used detection method is acid-fast staining. Different acid-fast staining methods have varying degrees of sensitivity and specificity and may require skilled technicians, such as a specialized microscopist, to perform the analysis. On the other hand, IF antibody staining is also frequently used to detect Cp. oocyst, but the non-specificity of the antibody due to the cross reaction with other species might be a problem and this method require large number of oocysts ranging from 50000-500,000 oocysts/g in order to give a positive detection feedback.

Flow cytometry feature higher sensitivity than acid-fast staining and IF antibody staining but it requires an expensive flow cytometry equipment in order to carry out the analysis. PCR also features a highly sensitive detection feedback which able to detect oocysts number ranging from ioo-ι,οοο oocysts/g. However, it is time consuming and the chemical reactants needed to carry out PCR are relatively expensive compared to the other detection methods. Therefore, there exists a more sensitive, accurate and specific but less time consuming and relatively inexpensive method and device for detecting the presence of Cp. parvum oocysts in a sample. Summary of the invention

In a preferred aspect of the present invention, there is provided a device for detecting the presence of a parasite in a sample, the device comprising: (a) a carbon substrate, wherein an antibody or an antibody fragment specific to an epitope of the parasite is immobilized on the substrate; and (b) an electrical circuit configured to monitor conductance across the substrate, wherein the presence or absence of the parasite is detected based on a change in the conductance.

By "electrical conductance", it is meant to include any passage of electric current across the carbon substrate.

Preferably, the carbon substrate is a graphene sheet.

Preferably, the parasite is Cryptosporidium. More preferably, the parasite is Cryptosporidium parvum. Still more preferably, the epitope is a Cryptosporidium parvum oocyst antigen.

Preferably, the concentration of the parasite is 25 oocysts/ml. Preferably, the antibody or antibody fragment is a monoclonal antibody.

Preferably, the antibody or antibody fragment is labelled with a fluorescent.

Preferably, the sample is a water sample selected from the group: recreational water, water reservoirs and biological sample.

In another preferred aspect of the present invention, there is provided a method of detecting the presence of a parasite in a sample, the method comprising: (a) contacting the sample with an antibody or an antibody fragment that binds specifically to an epitope of the parasite; and (b) detecting the presence of the parasite-bound antibody in the sample.

Preferably, the antibody or antibody fragment is immobilized on a carbon substrate. More preferably, the carbon substrate is a graphene sheet.

Preferably, the method further comprising applying an electric potential across the graphene sheet, wherein the presence of the parasite-bound antibody is detected by monitoring the change in the conductance across the graphene sheet.

Preferably, the parasite is Cryptosporidium. More preferably, the parasite is Cryptosporidium parvum. Still more preferably, the epitope is a Cryptosporidium parvum oocyst antigen. Preferably, the concentration of the parasite is 25 oocysts/ml.

Preferably, the antibody or antibody fragment is a monoclonal antibody.

Preferably, the antibody or antibody fragment is labelled with a fluorescent.

Preferably, the sample is a water sample selected from the group: recreational water, water reservoirs and biological sample.

Preferably, with regard to the both aspects of the invention set out above, the device comprises a graphene sheet configured wherein antibodies specific to a Cp. oocyst are immobilized on the graphene sheet. A sample containing Cp. oocysts are made to hybridize or bound to those antibodies bound on the graphene sheet. The graphene sheet is then congfigured as field-effect device for detecting Cp. oocysts by monitoring the drain current of the device. This is the first time using field-effect based biosensor for parasitic protozoan detection. The change in the graphene device drain current was caused by the specific binding of Cp. oocysts to the antibodies that were immobilized onto the graphene sheet surface. The presence (or absence) of Cp. oocysts in the sample were detected by shifting of conductance from the device. Preferably, the graphene sheet is a large-sized chemical vapor deposition (CVD) grown graphene sheet.

Preferably, the device comprises a plurality of graphene sheets. More preferably, the device comprises a plurality to microfluidic channels, wherein each channel is made of a graphene sheet. The antibodies specific to Cp. oocysts are bound to the surface of the graphene sheets.

The presence of Cp. oocysts causes the change in the transport characteristics of the antibody-functionalized graphene device, which can be measured in terms of the dependence of drain current on the sweep of the gate voltage or the real-time drain current data under a constant gate voltage. By "transport characteristics", it is meant to include the (a) change of the transfer curves, i.e. the dependence of drain current on the sweep of the gate voltage; or (b) real-time monitoring of the drain current change under a constant gate voltage. The exact transport characteristics are shown in Figures 9 to n described below.

Advantageously, the device of the present invention results in higher sensitivity, higher conductivity and larger detection area compared to the prior art of detecting Cryptosporidium parvum oocysts. In particular, but not limited to, the present invention of utilizing a graphene sheet provides the above mentioned advantageous over the use of a carbon-based devices.

Redox enzyme-modified chemical vapor deposition (CVD) grown graphene for glucose and glutamate detection and DNA hybridization detection were some examples of graphene-based biosensor. In view of recent progress in graphene-based biosensor, functionalized graphene is used as the active sensing material to detect the C. parvum at its oocysts stage of proliferation. Also, advantageously, the present invention may be used to detect other microorganisms, for e.g. bacterial, viral, protozoan so long as the chosen antibody is able to bind specifically to the targeted micro-organisms. Depending on the micro-organism in question, any such suitable antibodies may be used to bind to the graphene sheet. Brief description of the figures

In order that the present invention may be fully understood and readily put into practical effect, there shall now be described by way of non-limitative examples only preferred embodiments of the present invention, the description being with reference to the accompanying illustrative figures.

In the Figures: Figure l is a photo of the graphene sheet with electrodes according to an embodiment of the present invention;

Figure 2 is a Raman spectrum showing mono-layered and few-layered graphene domains of the graphene sheet according to an embodiment of the present invention;

Figure 3 is a photo of an Atomic Force Microscopy showing the topography of the graphene sheet according to an embodiment of the present invention;

Figure 4 is a schematic drawing showing the non-covalent immobilization of anti-bodies on the surface of the graphene sheet according to an embodiment of the present invention;

Figure 5 is a super-imposed photo of a fluorescence microscopy image and optical microscopy image of the graphene sheet according to an embodiment of the present invention;

Figure 6 is a schematic drawing of the device according to an embodiment of the present invention; Figure 7 is a photo of the device according to an embodiment of the present invention;

Figures 8(a) and 8(b) are a SEM image and fluorescence microscopy image of a single Cp. oocyst (scale bar = 2 μιη); Figure 9 is a graph showing electrical detection in response to various concentrations of Cp. oocysts;

Figure 10 is the binding curve according showing the concentration dependence of the change in drain current (Δ Id) at Vg=-o.72V;

Figures 11(a), 11(b) and 11(c) are graphs showing real-time Cp. oocyst sensing with high sensitivity and specificity - (a) Id vs. time during real-time sensing: solutions delivered into the PDMS channel are firstly buffer, then 10², 10³, and 10⁴ Cp. oocyst per 4mL solutions,

(b) the binding curve according showing the concentration dependence of the change in drain current (Δ Id) at Vg=-o.6oV,

(c) Control sensing experiment against Giardia lamblia cysts: the orange arrow marked the time when the solution was switched from buffer to 10⁵ Giardia lamblia cysts per 4mL solution, and no significant conductance change was observed;

Figure 12 is a SEM image of the presence of Cp. oocysts on the graphene sheet according to an embodiment of the present invention after electrical measurement;

Figure 13 shows the transfer curves of the graphene field-effect device according to an embodiment of the present invention after functionalization; Figure 14 shows the transfer curves in response to Cp. oocyst solutions of different concentrations vs functionalized/non-functionalization of the device; and

Figure 15 is a table showing a comparison of the transfer curve characteristics of the non- functionalized device vs. the functionalized device in response to Cp. oocyst solutions of different concentrations.

Detailed description of the preferred embodiments The present invention relates to the use of a graphene sheet, in particular a chemical vapor deposition (CVD) grown grapheme sheet that is functionalized, configured and adapted for placement in a device for detecting the presence or absence of C. parvum oocysts (Cp. oocysts) by monitoring the conductance of the device. The presence of Cp. oocysts is detected by shifting of conductance from the graphene sheet when these oocysts are bound specifically to the antibody reagent immobilized onto the graphene sheet.

From experiment results obtained, the present invention features high sensitivity and almost instant detection feedback for Cp. oocysts. In particular, Cp. oocysts as small as 25 oocysts/ml suspended in a PBS solution can be detected. As such, it is demonstrated that graphene is a potential candidate for use in a device for the detection of the Cp. oocysts. Preparing and fabricating the graphene substrate

Carbon substrates include carbon-based nanomaterials such as nano diamond, carbon nanotube, and graphene have been popularized due to their unique chemical and physical properties. Graphene is a 2D sp2-hybridized carbon sheet with one-atom thickness. Generally, by "graphene", it meant to include any thin film material which has a thickness of several nanometers and in which carbon atoms are two-dimensionally aligned. Because of its unique structure and special properties, graphene has attracted increasing attention in recent years. Its high theoretical surface area (2630 m²g^_1), chemically stability and high electrical conductivity make it an attractive material for applications in nanoelectronics, optoelectronics, energy-storage systems and chemical sensors.

In preparing the graphene sheet of the present invention, a mixture of methane and hydrogen was used as the carbon source to grow graphene substrate or sheet with single- layered domains on a Ni film (~500 nm thick, evaporated on Si0₂ / Si wafer) by utilizing chemical vapor deposition (CVD) method at about iooo°C. After that, poly(methyl methacrylate) (PMMA) which dissolved in chlorobenzene was spin-coated on the as- grown graphene, followed by baking at 120 °C for 20 min. Subsequently, the Ni film was etched away by the HCl solution (HC1 : H₂0 = 1 : 10) over a period of 8 h and the PMMA/graphene sheet was then put on a quartz substrate after deionized (DI) water rinsing.

After drying in air, a small amount of liquid PMMA / chlorobenzene solution was dropped onto it to dissolve the PMMA followed by acetone cleaning to remove PMMA. Finally, the PMMA/ graphene film on quartz substrate was annealed at 450°C for 20 min in H₂/Ar atmosphere to remove any remaining PMMA.

To fabricate the graphene field-effect device, layers of titanium (10 nm) and gold (40 nm) were evaporated to form the source and drain electrodes at the two ends of the graphene sheet, which defined an active area of 3 x 3mm. Subsequently, a home-built flow cell (channel length of 3mm, width of lomra and height of imm) made from Polydimethylsiloxanes (PDMS, Dow Corning Sylgard 184) block was then placed on top of the device with silicone rubber (Dow Corning 3140 RTV coating) as the adhesive layer which isolated and protected the electrodes from contacting the test solutions.

The CVD-grown graphene substrate used for fabricating the present device was characterized with Raman spectroscopy and atomic force microscopy (AFM), and the results are shown in Figures 2 and 3, respectively. The mono-layered and few-layered graphene domains were verified by the characteristic G/2D Raman peaks. Figure 1 shows the arrangement of a graphene sheet 10 in contact with two separate electrodes 20 according to an embodiment of the present invention. Figure 7 shows a photo of the fabricated field-effect device 5 incorporating the graphene sheet sheet 10 and electrodes 20 as shown in Figure 1.

Functionalization - Immobilization of antibody on graphene substrate

In order to bio-functionalized the graphene sheet of the present invention, non-covalent bio-functionalization of the surface of the graphene sheet 10 substrate was carried out. The general steps are illustrated in Figure 4.

In particular, in carrying out the non-covalent bonding and immobilization of antibodies on the graphene sheet 10 substrate, the graphene sheet 10 substrate was incubated in a 5mM linker molecule 15 (in the present case, l-pyrenebutanoic acid succinimidyl ester, i- DNA Biotechnology) solution in dimethylformamide (DMF) for 2 hours at room temperature, and washed with pure DMF and DI water. The linker-modified graphene sheet 10 was then incubated with an antibody reagent (in the present case, A400FLR-1X obtained from Waterborne Inc.) overnight at 4°C. This antibody reagent consists of a fluorescein-labeled (bright apple green when viewed under a fluorescence microscope) mouse monoclonal antibody 25 made to oocyst outer wall antigenic sites (epitopes) of C. parvum. After the incubation, the device 5 was rinsed three times in DI water to wash away excess reagent. The antibody reagent is genus-specific and will bind only to the oocysts if they are present. Examples of linker molecules/antibodies that may be used include (a) l-pyrenebutanoic acid, succinimmidyl ester, and (b) Water borne Inc. A400FLR-1X. Other examples as would be known to the skilled person may be used.

In order to verify the immobilization of the antibodies onto the graphene sheet 10 surface, a fluorescence image was taken after the functionalization and Figure 5 shows the super-imposed fluorescence microscopy image on top of an optical microscopy image of the same device 5 area. The image showed that only the area with the graphene sheet 10 was immobilized with the fluorescein-labeled antibody reagents which appeared bright apple green, and that the quartz 30 substrate was not modified with the antibodies.

Carrying out the analysis of a sample

After functionalization of graphene sheet 10 (immobilisation of the antibodies), the device 5 may be used to test for Cp. oocysts immediately. However, a baseline value will need to establish first for the buffer solution to serve as a reference point in the testing.

A perspective view of the device 5 is shown in Figure 6. Figure 7 is a photo of an actual device. With reference to Figure 6, the device 5 comprises a quartz 30 substrate which serves as a supporting base on which the functionalized graphene sheet 10 is placed on. The functionalized graphene sheet 10 is placed between two electrodes - drain electrode 20(a) and source electrode 20(b). A further gate electrode 40 is connected to the device 5. In particular, the gate electrode 40 is connected to the fluid sample that is introduced into the device 5 for the detection of Cp. oocysts. The electrodes 20(a), 20(b) and 40 provide and measure voltage and/ or current in the device 5. Probe pins 65 connect to the electrodes 20(a) and 20(b) which measure conductance changes and/or current changes on the graphene sheet 10. The electrodes 20(a), 20(b) and 40 and probe pins 65 connect to a processor for the measurement of the electrical conductance across the graphene sheet 10. The processor may be a semiconductor parameter analyzer that can measure or provide either voltage or current to the electrodes and probe pins that are connected to it. The device 5 further comprises a flow cell 45 for placement above the graphene sheet 10. The flow cell 45 comprises an inlet 50 for allowing a sample to be introduced into the device 5, an outlet 55 for the sample to exit the device 5, and at least one fluidic channel 60 to delivering the sample to the surface of the graphene sheet 10. The fluidic channel 60 may introduce the sample to a reaction chamber in the device 5 which allows the sample to come into contact with the graphene sheet 10. In an alternative embodiment, there may be more than one fluidic channel 60 to deliver the sample to the surface of the graphene sheet 10 - each channel having its own electrodes that may be connected to an appropriate processor for monitoring current changes across the graphene sheet 10. The sample may be any sample obtained from a patient or a body of water. The sample may be treated first prior to being introduced into the device 5. The flow cell 45 may be a PDMS (Polydimethylsiloxane) flow cell that provides a controlled fluid environment for a sample containing Cp. oocysts to interact with the active sensing area, i.e. graphene sheet 10 of the device 5. The flow cell 45 may be placed over the quartz 30 that supports the graphene sheet 10. Silicone rubber (Dow Corning 3140 RTV coating) may be used as an adhesive layer between the flow cell 45 and the quartz 30 support in order to isolate and protect the electrodes 20(a), 20(b) from contacting the test sample. A pump (not shown in the Figure) may be used to feed the sample into the inlet 50 and through the device 5 in a controlled pre-determined flow rate and direction flow. In order to detect the presence or absence of Cp. oocysts in the sample fluid, the sample fluid may be allowed to incubate with the graphene sheet 10. An electric potential is then applied across the graphene sheet 10. A constant voltage or sweeping voltage may also be applied at the gate eletrode 40 depending on the detection methods. If Cp. oocysts are present in the fluid sample, the parasite-bound antibody complex will be detected via monitoring the change in the conductance across the graphene sheet 10. This conductance may be monitoring by a semiconductor parameter analyzer that is connected to the source, drain and gate electrodes 20(a), 20(b) and 40. The presence of Cp. oocysts can be detected by either (1) the change of the transfer curves, i.e., the dependence of drain current on the sweep of the gate voltage, or (2) realtime monitoring of the drain current change under a constant gate voltage. The best condition for (1) is :

Vs= oV , Vd=o.5V, sweeping Vg from lV to -lV, monitoring Id versus Vg.

The best condition for (2) is:

V_g = -0.6V, Vs= oV , Vd=o.5V and monitor Id versus time.

Figures 8(a) and 8(b) show SEM image and fluorescence microscopy images of a single Cp. oocyst (scale bar = 2 μπι).

For Cp. oocysts sensing tests, various concentrations of Cp. oocysts were delivered into the PDMS flow cell in buffer solutions consisting of PBS with antibiotics (penicillin, streptomycin and gentamicin), Amphotericin B, and 0.01% Tween 20, and the electrical measurement for the presence of Cp. oocysts was carried out. The solutions were delivered with a flow rate of 0.30 mL/h through the fluidic channel 60. The antibody is genus-specific and binds only to the oocysts if they are present.

Two detecting (or sensing) methods were used to characterize the change in the graphene field-effect device drain current in response to the presence of Cp. oocysts. In the first method, two minutes after each solution of the Cp. oocysts was delivered into the flow cell, the transfer curve was measured by sweeping the liquid gate voltage (V_g) from 1.0 V to -1.0 V with a Ag/AgCl wire in contact with the solution and monitoring the drain current (Id) while the source-drain voltage (V_Sd) was kept constant at 0.5 V. The transfer curves were then compared with each other to show the trend of drain current change. In the second method, real-time monitoring of the drain current Id under constant V_Sd and V_g was carried out as solutions of different concentrations of Cp. oocyst were delivered into the flow cell consecutively.

In the sensing experiment using the first method, the transfer curves of the functionalized graphene device in solutions containing various concentrations of Cp. oocyst were measured, and the data is plotted in Figure 9. The drain current of the present device exhibited ambipolar behavior as a function of the liquid gate voltage (V_g) applied to the solution. The initial ambipolar transfer curve measured in buffer solution without Cp. oocyst is shown in black in the figure, and as we delivered into the flow cell a series of increasing concentrations of Cp. oocyst from ios, io⁴, ios, to io⁶ (oocysts per 4mL), it was observed that the slope of both branches of the ambipolar transfer curve increased. This increasing slope also means that, at a constant V_g, the Id increases as the Cp. oocyst concentration increases, and this concentration dependence of the change in drain current (Δ Id) at a constant V_g (for example, when the liquid gate voltage V_g was swept to the voltage of -0.72V) is summarized in the binding curve shown in Figure 10. It can be seen from the binding curve that the dynamic range of Cp. oocyst sensing was ios -io⁶ (oocysts per 4mL), as the binding curve saturated at higher concentration range (around ios -10⁶). However, looking at the trend of the transfer curves in Figure 9, a sensitivity of 10² (oocysts per 4mL) could be achieved. In addition, it was also observed the unbinding signal of Cp. oocysts upon buffer washing after the sensing of Cp. Oocyst, shown as dotted grey line in Figure 9. The transfer curve after washing lied somewhere between those for Cp. oocyst with concentrations of 103 and 10⁴, indicating there were some Cp. oocysts that irreversibly adsorbed onto the graphene sheet.

In terms of the detecting and sensing mechanism of the present device, since both graphene and single-walled carbon nanotubes (SWNTs) are formed by sp2-bonded carbons, it is highly possible that the sensing mechanisms for the carbon-based materials are similar. Previously suggested mechanisms in SWNT sensors are electrostatic gating, changes in gate coupling, carrier mobility, and Schottky barrier effect. First, the electrostatic gating sensing mechanism by comparing the transfer curve change pattern of the non-functionalized versus the functionalized graphene field-effect devices in response to the Cp. oocysts in buffer solutions was examined. Figures 14(a) and 14(b) show the response of the pristine (i.e. graphene sheet without any immobilized antibodies) of graphene device to the Cp. oocysts in buffer solutions. All the transfer curves left-shifted, suggesting the Cp. oocysts adsorbed onto the graphene surface acted as a positive gate through the electrostatic gating mechanism. The shifts of transfer curves of the functionalized device were also examined, and Figure 14(c) shows the zoom-in of Figure 9 for this purpose. Comparing the transfer curve behaviors of the functionalized and the non-functionalized graphene devices, which is summarized in the table shown in Figure 15, the left-shift of the minimum conductance point for the functionalized device is far less significant than that of the non-functionalized device: for example, in response to the icH (count/4mL) Cp. oocysts in buffer solution, the minimum conductance point left-shifted by 0.17 V for the non-functionalized device, in contrast to only 0.031 V for the functionalized device. The reduced electrostatic gating effect in the case of the functionalized device compared to bare graphene, may be due to the presence of the antibody modification layer between the graphene surface and Cp. oocysts in the case of functionalized device. Secondly, upon the analysis of the slopes of both the n- and p- branches of the ambipolar transfer curve, it appears that the dominating sensing mechanism of the functionalized device is the change in the gate couple efficiency. As summarized in the table shown in Figure 15, in the functionalized device, the slope of both n- and p- branches increased significant as the concentration of Cp. oocysts increased, for example, an average of 73% increase in ios (counts/4mL) Cp. Oocyst solution, compared to an average of 25% increase in the non-functionalized device for the same Cp. oocyst concentration. Further work is necessary to elucidate the details on how the binding of Cp. oocysts to the antibody modification layer on the graphene surface changed the electrochemical double-layer capacitance of the graphene- liquid interface. Thirdly, considering the fact that the electrodes/graphene contact junctions have all been passivated with PDMS from any exposure to solutions, the Schottky barrier mechanism is ruled out here. Lastly, the change in carrier mobility is unlikely to explain the sensing mechanism because the conductance in both the p- and n- branches was observed to increase whereas the mobility mechanism predicts a conductance decrease as the adding of Cp. oocysts is expected to introduce more carrier scattering centers and hence reduce carrier mobility. In summary, based on the results shown, it can be concluded that the underlying sensing mechanism of of the present graphene biosensor device is not simply the effect of electrostatic gating but is mainly due to the interfacial capacitance change.

Besides the detection of Cp. oocyst with the first method of comparing transfer curves (sweeping liquid gate Vg while keeping Vsd constant) under different target concentrations, the second method was also tested - monitoring in real time the steplike response of the device current (with constant Vs_d and V_g) to different target concentrations. It is worth pointing out that the experimental results in Figure 9 demonstrated the importance of choosing the liquid gate potential V_g in the second method: depending on the value of V_g that was applied during a real-time sensing experiment of Cp. oocyst, the magnitude and even sign of the Id change can vary. V_g = - 0.6V was chosen to carry out the real-time sensing experiment using another functionalized graphene device with similar transport performance, and the results were shown in Figure il(a). The Id recorded in the first 366s was the baseline value when the graphene device was in contact with buffer solution. All the buffer solutions used in this real-time sensing experiment were ten times diluted from the one that were used for the transfer curve measurements. The Debye length is longer in diluted buffer, so the sensitivity of the graphene field-effect device could be pushed further by decreasing the screening effect of ions. The first arrow A in Figure 11(a) marked the time when the rear end of the tubing leading to the PDMS channel was switched from connecting with pure buffer to the solutions of 10² Cp. oocyst per 4mL buffer. The conductance after the switching point fluctuated for ~iooos and then showed a more significant increase and reached a steady plateau. The measurement of the Id was interrupted for a while due to the limitation of our data recording system. Nevertheless, it was clearly shown that the presence of Cp. oocyst in the solution caused an increase in the drain current, and this was consistent with what was observed in the first sensing method with the transfer curve measurement. The second and third arrows B and C in Figure 11(a) marked the time that the tubing was switched to the solutions of io3 and then 10⁴ Cp. oocyst per 4mL buffer. As the concentration of the Cp. oocyst increased, the conductance showed a sharper increase in a shorter time compared to its response to 10² Cp. oocyst per 4mL buffer. Figure 11(b) summarized the concentration dependence of the change in drain current ( Δ Id) when the liquid gate voltage V_g was at -0.6 V. This sensing experiment showed the feasibility of rapid detection of Cp. oocyst in a liquid sample within i5~30min, and the consumption of sample volume was only sub-milliliter. The device demonstrated very high sensitivity by showing clear signals to Cp. oocyst solution with concentration down to 10² per 4mL solution (which is about 25 counts/gram) as well as high selectivity by showing no response to ios Giardia lamblia cysts per 4mL solution, another pathogenic protozoa commonly present in water (shown in Figure 11(c)). In addition, scanning electron microscopy (SEM) images were taken in order to verify the presence of Cp. oocysts after the electrical signal was measured. Figure 12 showed that Cp. oocysts were observed on the graphene sheet. All SEM images were taken after the electrical measurement, and the device was rinsed 3 times using DI water and air dried before platinum was sputtered on it for SEM observation. No Cp. oocysts were observed on the area outside of the graphene sheet. All electrical measurement were conducted using Keithley 4200 semiconductor characterization system (e.g. a parameter analyzer) at room temperature. The transport properties of the graphene field-effect device after biofunctionalization were measured by sweeping the liquid gate voltage Vg from 1.0 V to -1.0 V, as shown in Figure 13. The liquid gate voltage was applied through a Ag/AgCl wire that was in contact with the buffer solution covering the device. In order to confirm the stability of the graphene field-effect biosensor, three additional sweeps were carried out after the first transport measurement. The transfer curves overlaid on each other and there was no obvious shape change for various Vg sweeps, which confirmed the good stability of the device. Moreover, only devices that could reproduce stable and repeatable Id-Vg curves were chosen for sensing experiments.

In summary, large-size CVD grown graphene sheets configured as field-effect devices for sensing of Cp. oocysts with high sensitivity and specificity was demonstrated in this work. After the functionalization of the graphene device with the linker molecule and immobilization of specific antibody, the presence of Cp. oocysts can be detected by either (1) the change of the transfer curves, i.e., the dependence of drain current on the sweep of the gate voltage, or (2) real-time monitoring of the drain current change under a constant gate voltage. This study demonstrated the graphene field-effect device as a promising candidate for the rapid detection of the Cp. oocysts with high sensitivity and specificity, and the biofunctionalized graphene device platform can be applied in the sensing of other bacterial, viral, protozoan waterborne pathogens and play important roles in water quality control.

Similar configuration and strategy can be used to detect Cp. oocysts by changing the graphene sheet to other functionalizable material, for e.g. Si nanowires, MoS₂ sheet etc. Recently, we had shown that it is possible to decorate MoS₂ sheet with Au nanoparticle and this open up possibility on making biosensors based on Au nanoparticle modified MoS₂ sheet. However, the functionalization scheme will be differed from the previous case. In order for this material to work for Cp. oocysts detection, the Au nanoparticle decorated MoS₂ sheet need to functionalized with 1.5 mM 3,3'-dithio-bis(propionic acid N- hydroxysuccinimide ester) (DTSP, Sigma Aldrich) in dry dimethylsulfoxide (DMSO) solution for 12-24 h in dark and followed by extensive rinsing in DMSO. After that, antibody was then coupled to the succinimidyl(NHS)-terminated Au surface for a period of 2-4 h. Besides from the functionalization scheme, the other configuration and testing should be similar to the above mentioned case. Whilst there has been described in the foregoing description preferred embodiments of the present invention, it will be understood by those skilled in the technology concerned that many variations or modifications in details of design or construction may be made without departing from the present invention.

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Claims

Claims

1. A device for detecting the presence of a parasite in a sample, the device comprising:

(a) a carbon substrate, wherein an antibody or an antibody fragment specific to an epitope of the parasite is immobilized on the substrate; and

Ob) an electrical circuit configured to monitor conductance across the substrate,

wherein the presence or absence of the parasite is detected based on a change in the conductance.

2. The device according to claim l, wherein the carbon substrate is a graphene sheet.

3. The device according to any one of claims 1 or 2, wherein the parasite is Cryptosporidium.

4. The device according to claim 3, wherein the parasite is Cryptosporidium parvum.

5. The device according to any one of the preceding claims, wherein the epitope is a Cryptosporidium parvum oocyst antigen.

6. The device according to claim 5, wherein the concentration of the parasite is 25 oocysts/ml.

7. The device according to any one of the preceding claims, wherein the antibody or antibody fragment is a monoclonal antibody.

8. The device according to any one of the preceding claims, wherein the antibody or antibody fragment is labelled with a fluorescent.

9. The device according to any one of the preceding claims, wherein the sample is a water sample selected from the group: recreational water, water reservoirs and biological sample.

10. A method of detecting the presence of a parasite in a sample, the method comprising:

(a) contacting the sample with an antibody or an antibody fragment that binds specifically to an epitope of the parasite; and

(b) detecting the presence of the parasite-bound antibody in the sample.

11. The method according to claim 9, wherein the antibody or antibody fragment is immobilized on a carbon substrate.

12. The method according to claim 10, wherein the carbon substrate is a graphene sheet.

13. The method according to claim 11, further comprising applying an electric potential across the graphene sheet, wherein the presence of the parasite-bound antibody is detected by monitoring the change in the conductance across the graphene sheet.

14. The method according to any one of claims 9 to 12, wherein the parasite is Cryptosporidium.

15. The method according to any one of claims 9 to 13, wherein the parasite is Cryptosporidium parvum.

16. The method according to any one of claims 9 to 14, wherein the epitope is a Cryptosporidium parvum oocyst antigen.

17. The method according to claim 16, wherein the concentration of the parasite is 25 oocysts/ml.

18. The method according to any one of claims 9 to 15, wherein the antibody or antibody fragment is a monoclonal antibody.

19. The method according to any one of claims 9 to 16, wherein the antibody or antibody fragment is labelled with a fluorescent.

20. The device according to any one of the preceding claims, wherein the sample is a water sample selected from the group: recreational water, water reservoirs and biological sample.