WO2014081393A1

WO2014081393A1 - Method for detection or an antigen

Info

Publication number: WO2014081393A1
Application number: PCT/SG2013/000494
Authority: WO
Inventors: Kam Man Hui; Yafeng WU; Yuejun Kang
Original assignee: Singapore Health Services Pte Ltd; Nanyang Technological University
Priority date: 2012-11-21
Filing date: 2013-11-21
Publication date: 2014-05-30

Abstract

The present invention relates to methods of detection. More particularly, methods of determining the presence of at least one antigen in a sample, and an apparatus and a kit for use in said method. The method involves using nanoparticle compositions, each comprising a nanoparticle, a plurality of quantum dots and an antibody, together with a graphene oxide-coated electrode attached to a plurality of antibodies, the graphene oxide coating on the electrode has been electrochemically reduced. Said antibody and antibodies being capable of binding to the at least one antigen. Contacting the sample with the electrode and contacting the electrode with the nanoparticle compositions and subsequently detecting the presence of the at least one antigen in the sample by detecting at least one property of each of the pluralities of quantum dots bound to the electrode by way of the at least one antigen, at least one third antibody and at least one first or second antibody.

Description

METHOD FOR DETECTION OF AN ANTIGEN

Field of the invention

The present invention relates to methods of detection. More particularly, methods of determining the presence of at least one antigen in a sample, and an apparatus and a kit for use in said method.

Background Metastasis is the primary cause of mortality in most cancer patients and it is initiated by cancer cells that are shed and transported through the circulation from the primary tumor to distant organs. It has been estimated, using an experimental rat model system, that as many as 3-4x10⁶ mammary carcinoma cells can be shed every day into the circulation from a primary tumor weighing a gram, though few of these ever form metastases. It nevertheless holds that the number of metastases formed is generally proportional to the number of tumor cells shed into the circulation and therefore it has been suggested that circulating tumor cells (CTC) can be used as a noninvasive measure to monitor treatment efficacy and disease progression or recurrence. However, even if one assumes that there is no clearance of the tumor cells shed into the circulation, one would have a maximum estimation of 200 tumor cells mL^"1 of blood for an average individual with approximately 5 L of blood. This concentration equates to about 0.004% of all the white cells in the blood and hence, poses key technical challenges for the detection and quantification of these low-abundance CTCs due to the high sensitivity and specificity needed for these methodologies. Currently employed strategies rely on either the physical properties, expression of biomarkers, or the functional characteristics of CTCs, including polymerase chain reaction (PCR)-based analysis, cytometric analysis and cell- enrichment steps to boost sensitivity. Although each of these methods represents significant technological advances and provides a basis from which to anticipate ongoing technological developments, none of these current approaches constitute the optimal platform for CTC isolation. These methods are expensive, time-consuming and of low specificity, and require advanced instrumentation and enrichment of the target cells in the sample. Moreover, assays relating to the enumeration of CTC typically remain limited by the inability to elute the captured cells for further molecular analysis. However, it is the molecular characterization of CTCs that offers confirmation and offers potential insights into the metastasis process.

Great efforts have been made toward signal amplification to significantly enhance the sensitivity for biomarker detection, including the employment of new redox-active probes; the integration of enzyme-assisted signal amplification processes; and the incorporation of nanomaterials to increase the upload of electrochemical tags, etc. The latter approach is particularly effective as it introduces multiple redox species per binding event. Silica nanoparticles (Si0₂) are the most commonly used nanoparticles in electrochemical immunosensors due to its unique features of monodispersed size, uniform structure, good biocompatibility and easy functionalization. QD-based electrochemical bioassay has received particular attention because of inherent miniaturization, low detection limits, low cost, and low power requirement. The high fluorescence quantum yield (QY) and high optical stability of QDs in comparison with other alternatives, such as organic fluorescent molecules and phosphorescent dyes, make them highly promising nanomaterials for photovoltaic cells, biosensors and light-emitting diodes.

Chen ei a/., Clin. Chim. Acta 2010, 411 , 1969-1975, described the use of CdTe QDs coated Si0₂ as a signal amplification method in latent membrane protein 1 (LMP-1 ) detection. Anti-LMP-1/QDs-coated Si0₂ nanoparticles were used as a label, which were introduced to the electrode surface through two immunoreactions. A detection limit of 1 pg mL^"1 was achieved, and the signal was amplified 2.8 times compared with traditional immunoassay. Qian ef a/., Biosens. Bioelectron. 2011 , 28, 314-319, have also proposed an electrochemical immunosensor based on CdSe and PbS QDs coated Si0₂, as dual labels for IgG and CEA determination. These labels were introduced to the surface of gold substrates through a subsequent sandwich immunoreaction, which allowed simultaneous detection of IgG and CEA. The lowest detectable concentration achieved was 3 pg mL^'1 for IgG and 5 pg mL^"1 for CEA, yielding a higher sensitivity than most traditional sandwich immunoassays.

There remains a need for alternative sensitive and specific assays for cells, in particular quantitative and qualitative assays that can detect and/or measure antigens on CTCs at low concentrations in a sample, and for assays that enable the elution of bound tumor cells. Summary of the Invention

The present invention addresses some of the problems in the art and provides a method for determining the presence of at least one antigen in a sample.

According to a first aspect, the present invention provides a method for detecting at least one antigen in a sample, comprising:

(i) providing a first nanoparticle composition comprising at least a first nanoparticle, a first plurality of quantum dots and a first antibody, where the first antibody is capable of binding to the at least one antigen and where the first plurality of quantum dots and the first antibody are attached to the first nanoparticle;

(ii) providing a second nanoparticle composition comprising at least a second nanoparticle, a second plurality of quantum dots and a second antibody, where the second antibody is capable of binding to the at least one antigen and where the second plurality of quantum dots and the second antibody are attached to the second nanoparticle;

(iii) providing a graphene oxide-coated electrode, the electrode being attached to a plurality of a„third antibody capable of binding to the at least one antigen ;

(iv) contacting the sample with the electrode;

(v) contacting the electrode with the first nanoparticle composition and the second nanoparticle composition; and

(vi) subsequently detecting the presence of the at least one antigen in the sample by detecting at least one property of each of the first and second pluralities of quantum dots bound to the electrode by way of the at least one antigen, at least one third antibody and at least one first or second antibody, wherein

the first plurality of quantum dots differs from the second plurality of quantum dots and the first antibody differs from the second antibody; and

the graphene oxide coating on the electrode has been electrochemically reduced.

Step (iv) may further comprise removing unbound sample from the electrode after contacting the sample with the electrode and/or (v) may further comprise removing unbound nanoparticle compositions from the electrode after contacting the electrode with the first nanoparticle composition and the second nanoparticle composition.

In one aspect there is provided an apparatus for use in the method of the invention, comprising a graphene oxide-coated electrode and a plurality of a third antibody capable of binding to at least one antigen, the plurality of the third antibody being attached to the electrode and the graphene oxide coating on the electrode having been electrochemically reduced.

In a further aspect, there is provided a kit of parts for use in the method of the invention, comprising:

the apparatus of the invention;

a first nanoparticle composition, comprising a first nanoparticle, a first plurality of quantum dots and a first antibody, where the first antibody is capable of binding to at least one antigen and where the first plurality of quantum dots and the first antibody are attached to the first nanoparticle; and

a second nanoparticle composition, comprising a second nanoparticle, a second plurality of quantum dots and a second antibody, where the second antibody is capable of binding to the at least one antigen and where the second plurality of quantum dots and the second antibody are attached to the second nanoparticle, wherein

the first plurality of quantum dots differs from the second plurality of quantum dots and the first antibody differs from the second antibody.

Brief Description of the Figures

The invention will be described herein by reference to the following figures.

Figure 1 illustrates the (A) preparation of various different Quantum Dot (QD)-coated Silica nanoparticles with attached antibodies, denoted as Si/Zn/anti-GPC3, Si/Cd1/anti-EpCAM, Si/Cd2/anti-GPC3; (B) fluorescent and electrochemical detection procedure of circulating tumor cells captured via Anti-EpCAM antibodies immobilized on an electrochemically reduced Graphene Oxide (GO)-coated Glassy Carbon Electrode (GCE).

Figure 2 shows results of (A) Electrochemical impedance spectroscopy of (a) chitosan/GCE, (b) chitosan/GO/GCE, and (c) chitosan/electrochemically reduced GO/GCE in 0.1 M KCI containing 5 mM Fe(CN)₆ ^2~/3~; (B) Records of anodic stripping voltammograms after the anti-EpCAM/chitosan/electrochemically reduced GO/GCE was incubated in 1 ^χ 10⁶ cells mL^"1 Hep3B cell solution for 30 min, and then in (a) Si/Cd/anti-EpCAM and Si/Zn/anti-GPC3 solution or in (b) CdTe QD-labeled anti-EpCAM and ZnSe QD-labeled anti-GPC3 for 50 min. Records of anodic stripping voltammograms after the anti- EpCAM/chitosan/GCE were incubated in 1 ^χ 10⁶ cells mL^"1 Hep3B cell solution for 30 min, and then in (c) Si/Cd/anti-EpCAM and Si/Zn/anti-GPC3 solution for 50 min. Records of anodic stripping voltammograms after the anti-EpCAM/chitosan/electrochemically reduced GO/GCE was incubated in (d) PBS solution without a cell or (e) PP5 cell solution. Records of anodic stripping voltammograms after the anti-EpCAM/chitosan/electrochemically reduced GO/GCE was incubated in 1 * 10⁶ cells ml_^"1 MCF-7 cell solution for 30 min, and then in (f) Si/Cd/anti-EpCAM and Si/Zn/anti-GPC3 solution for 50 min.

Figure 3 shows results of (A) Square Wave Voltammetry (SWV) of Si/Cd1/anti-EpCAM- Hep3B-anti-EpCAM/chitosan/ electrochemically reduced GO/GCE at Hep3B cell concentration of: 5, 10, 100, 500, 1000, 1 *10⁴, 5*10⁴, 1 *10⁵, 5><10⁵, 1 *10⁶ cells ml^"1; (B) Plot of peak current obtained by dissolved Si/Cd1/anti-EpCAM-Hep3B-anti- EpCAM/chitosan/ electrochemically reduced GO/GGE versus Hep3B cell concentration in incubation solution. Inset in (B): linear regression plot. Figure 4 shows results of (A) SWV of Si/Zn/anti-GPC3-Hep3B-anti-EpCAM/ chitosan/electrochemically reduced GO/GCE at Hep3B cell concentration of: 10, 100, 500, 1000, 1 *10⁴, 5*10⁴, 1*10⁵, 5x10⁵, 1 *10⁶ cells mL ¹; (B) Plot of peak current obtained by dissolved Si/Zn/anti-GPC3-Hep3B-anti-EpCAM/chitosan/electrochemically reduced GO/GCE versus Hep3B cell concentration in incubation solution. Inset in (B): linear regression plot.

Figure 5 shows (A) Fluorescence microscopic images of anti-EpCAM-coupled red nanobioprobes. (B) Fluorescence microscopic images of anti-GPC3-coupled green nanobioprobes. (C-F) Fluorescence microscopic images of Hep3B cells incubated with EpCAM-modified GCE, followed by incubating with Si/Cd1/anti-EpCAM and Si/Cd2/anti- GPC3 nanobioprobes. (C) Blue fluorescence from Hoechst 33342 localized in the nuclei. (D) Red fluorescence from Si/Cd1/anti-EpCAM nanobioprobes. (E) Green fluorescence from Si/Cd2/anti-GPC3 nanobioprobes. (F) A merged image of (C), (D), and (E). Here, each fluorescent dot came from a single nanobioprobe.

Figure 6. shows (A-C) Fluorescence microscopic images of MCF-7 cells (EpCAM positive, GPC3 negative) incubated with EpCAM-modified GCE, followed by incubating with Si/Cd1/anti-EpCAM and Si/Cd2/anti-GPC3 nanobioprobes. (A) Blue fluorescence from Hoechst 33342 localized in the nuclei. (B) Red fluorescence from Si/Cd1/anti-EpCAM nanobioprobes. (C) A merged image of (A) and (B). (D) Negative control. PP5 cells (EpCAM and GPC3 negative) incubated with EpCAM-modified GCE, followed by incubation with Si/Cd1/anti-EpCAM and Si/Cd2/anti-GPC3 nanobioprobes. No fluorescence could be detected.

Figure 7 shows TEM images of the as-synthesized (A) Si0₂ , (B) Si/Cd and (C) Si/Zn nanoparticles.

Figure 8 shows (A) XPS spectra of the as-synthesized CdTe QDs coated silica nanoparticles before (a) and after (b) further coupling with anti-EpCAM; (B,C) XPS spectra of the as-synthesized ZnSe QDs coated silica nanoparticles before (a) and after (b) further coupling with anti-GPC3.

Figure 9 shows (A) Anodic stripping voltammograms in the sandwich immunoassay as a function of incubation time during Hep3B cell capturing; (B) Anodic stripping voltammograms in the sandwich immunoassay as a function of incubation time during Ab- coupling nanobioprobe capturing. The concentration of Hep3B cell was 1 *10⁶ cells ml.^"1.

Detailed Description of the Embodiments The present invention relates to a method for determining the presence of at least one antigen in a sample.

We disclose herein the combination of quantum dots-coated silica nanoparticles with graphene-accelerated electron transfer to develop a dual signal amplification strategy producing an ultrasensitive and highly specific electrochemical and fluorescent protocol for the simultaneous measurement of two different types of protein biomarkers on the surface of tumor cells. CdTe- and ZnSe-coated silica nanoparticles, having uniform size distribution and good stability, could easily serve as tracing tags to label anti-EpCAM and anti-GPC3 (Figure 1A). Graphene oxide was immobilized on an immunosensor surface using chitosan and electrochemically reduced to accelerate electron transfer. The amino group on the chitosan film enabled the covalent attachment of the capture antibody (Figure 1 B). After sandwich-type immunoreaction, the two tracers were co-immobilized on the cell surface. Additionally, the binding between antibodies and tumor cells is non-covalent and thus offers the potential to elute the bound tumor cells, regenerating the immunosensor. The present design enabled a detection limit down to 10 cells mL^"1 and exhibited good stability, precision and accuracy, demonstrating its wide potential applications for clinical diagnostics.

Accordingly, in a first aspect there is provided a method for detecting at least one antigen in a sample, comprising:

(iii) providing a graphene oxide-coated electrode, the electrode being attached to a plurality of a third antibody capable of binding to the at least one antigen ;

(iv) contacting the sample with the electrode;

the graphene oxide coating on the electrode has been electrochemically reduced.

In step (vi), the first and second pluralities of quantum dots bound to the electrode are bound in the sense that they are respectively attached to first and second nanoparticles, which are each attached to their respective first and second antibodies. The first and second antibodies are bound to the at least one antigen, which is in turn bound to the electrode via at least one third antibody attached to the electrode.

This method is able to achieve a high level of sensitivity that meets the requirement for clinical applications. For example, when used to detect Hep3B cells in a sample using Anti- GPC3 and Anti-EpCAM antibodies in the above method, detection of 10 cells or fewer per ml_ of sample was shown to be feasible. For sensitive detection, the graphene oxide may preferably be graphene oxide that has been homogeneously coated onto the electrode, e.g. exfoliated graphene oxide. The graphene oxide may be electrochemically reduced after coating onto the electrode. The steps (i), (ii), (iii), (iv), (v) and (vi) may be performed in numerical order, but the order may be changed, provided that step (v) comes after step (iv) and before step (vi), and step (vi) is always performed last. For example, one or more of steps (i) to (vi) may be performed concurrently, or with overlap. Step (iv) may further comprise removing unbound sample from the electrode after contacting the sample with the electrode and/or (v) may further comprise removing unbound nanoparticle compositions from the electrode after contacting the electrode with the first nanoparticle composition and the second nanoparticle composition. When used herein, "detecting" refers to qualitative and/or quantitative detection and/or determination using indicative data. "Detecting" may, for example, relate to detecting the presence or absence of an antigen or group of antigens in a sample, measuring the amount of one or more antigens present in a sample, and also using such data to determine the presence or absence of a target cell. "Detecting" may also relate to detecting fluorescence from one or more quantum dots, measuring the intensity of the fluorescence from said quantum dots, detecting a peak of oxidation current at a particular voltammetric voltage, measuring the oxidation current at that voltage, etc..

The term "antigen" when used herein refers to a substance that is bound specifically by an antibody, a group thereof, or fragment thereof, via one or more epitopes on the antigen. For a given cell type having two or more different antigens specific to that cell type, the presence of these antigens may be indicative of the presence of that cell type in a sample, especially a sample of cells. Accordingly, "antigen/cell" is used to refer to an antigen and/or a cell and/or a cell fragment comprising the antigen, where applicable.

The term 'comprising' as used in the context of the invention refers to where the various r

compounds, components, ingredients, or steps, can be conjointly employed in practicing the present invention. Accordingly, the term 'comprising' encompasses the more restrictive terms 'consisting essentially of, 'consisting of and 'having', which may all be used to replace each instance of comprising and each other. With the term 'consisting essentially of it is understood that the method of the present invention 'substantially' comprises the indicated compound, component, ingredient or step as 'essential' element.

"Nanoparticle" generally refers to a particle having a diameter of about 1 to about 1000 nanometres (nm), and may be used to describe any particle which is dimensioned appropriately to bind to a target cell via an antibody. For example a nanoparticle may have a diameter of about 500 nm. Preferably the nanoparticle is small enough to allow for a plurality of nanoparticles to bind to the target cell, while also maximizing loading of quantum dots onto the nanoparticles to allow efficient signal amplification for detection of the cell. Suitable nanoparticles may have diameters of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 nanometres. For example, the size of the nanoparticles used in the examples below is about 100 nm in diameter. Nanoparticles of this size range have low steric hindrance, i.e. allowing for more nanoparticles to contact and potentially bind to the antigen/cell, and allow for high loading of signal molecules, i.e. quantum dots. Smaller nanoparticles have even lower steric hindrance but have a smaller surface area which limits the total number of signal molecules per particle; larger nanoparticles can load more signal molecules due to large surface area, however they have much higher steric hindrance. Particular nanoparticles may have diameters less than or equal to 100 nm. In certain cases, the nanoparticles may have larger diameters, e.g. 300-1000 nanometres, if this does not result in too much steric hindrance.

"Nanoparticle composition" refers to any composition comprising at least one nanoparticle. For example a carrier fluid or solvent may be included in the composition to disperse a plurality of nanoparticles, if desired or necessary. Such dispersion could then allow the nanoparticles to efficiently contact cells in a sample.

When used herein, "attached" refers generally to any form of coupling between two objects, e.g. a hydrogen bond, a covalent bond, ionic attraction, etc. The coupling may be direct or indirect, for example an antibody may be attached to an electrode indirectly via an interface material, the antibody being covalently bonded to the interface material.

The term "binding" as used in this specification, unless otherwise specified, only refers to antibody binding. In other words, it refers to the binding between an antibody and an antigen (or set of epitopes) for which the antibody has an affinity to bind to. "Antibody" as used in this specification refers generally to any naturally-occurring or artificial antibody or part thereof, for example a monoclonal antibody, polyclonal antibody, antibody fragment, that is capable of binding to an antigen. As a skilled person will appreciate, some antibodies work in groups to bind certain antigens and therefore "antibody" also refers to such groups of antibodies or fragments thereof. Two antibodies binding to two different antigens, e.g. two different parts of a cell, are therefore considered two different antibodies.

When used herein, "quantum dot" refers to a nanocrystal typically made of semiconductor material (for example silicon, cadmium selenide, cadmium sulfide, indium arsenide, etc.). Because of their small size, quantum dots display optical and electrical properties that are different from those of the corresponding bulk material. For example, they emit photons under excitation, at a particular wavelength depending on the size and/or the band gap of the quantum dot. Accordingly, two quantum dots made of the same material may be considered different because they emit light at different wavelengths. As a skilled person will appreciate, some types of quantum dots comprise a core/shell structure allowing the use of other combinations of materials (e.g. CulnS/ZnS core/shell quantum dots). Some quantum dots comprise polymer coatings to enhance and/or modify their properties, e.g. enhanced stability, etc.

The phrases "at least a ..." and "a ..." refer to one or more of the object being described. For example, "at least a first nanoparticle" and "a first nanoparticle" may refer to one or more of the first nanoparticle.

The detection of at least one property of each of the first and second pluralities of quantum dots may comprise dissolving the first and second pluralities of quantum dots to obtain metal ions and performing voltammetric analysis on the metal ions to determine the identity and/or amount of the first and second pluralities of quantum dots. The voltammetric analysis may comprise Square wave stripping voltammetry. Since the quantum dots being dissolved are those that were bound by their respective attached antibodies to the antigens/cells captured by the plurality of the third antibody on the electrode, the metal ions resulting from their dissolution can be identified and/or correlated to the amount of first and second pluralities of quantum dots bound. This in turn can provide information about the antigens in the sample which were bound by the electrode and first and second antibodies. This information may be useful, e.g. to determine whether a target cell type is present in a sample, using antibodies specific to antigens that are indicative of that cell type. Further, the amount of bound antigen from a sample can be used to determine the concentration of the target cell type in the sample.

The detection of at least one property of each of the first and second pluralities of quantum dots may additionally or alternatively comprise detecting the fluorescence of the first and second pluralities of quantum dots. In some situations, this may be helpful to confirm that the antigens being detected represent a desired substance. For example, if whole cells are desired and the antigens are distributed within a whole cell in a known pattern, detecting the fluorescence from the quantum dots on the bound nanoparticles can confirm that whole cells have been bound to the electrode.

The at least one antigen may comprise at least two antigens specific to a target cell, wherein the presence of the at least two antigens is indicative of the presence of the target cell. The method may be useful for multiplexed detection of more than one set of antigens that may be specific to different target cells. For example, the at least one antigen may further comprise at least two additional antigens specific to at least one additional target cell, wherein the presence of the at least two additional antigens is indicative of the presence of the at least one additional target cell. The method may be advantageous in that compared to other detection methods where the cells are destroyed or damaged, it may be possible to maintain the bound cells without damage on the electrode, to be eluted as and when needed from the electrode, e.g. for further analysis by methods such as molecular characterization of CTCs that offers confirmation and potential insights into the metastasis process. Accordingly, the method may further comprise a step of eluting the bound target cell from the electrode, optionally including a step of eluting bound nanoparticles from the target cell, and/or the method according to claim 6 further comprising a step of eluting the bound at least one additional target cell from the electrode, optionally including a step of eluting bound nanoparticles from the at least one additional target cell. The target cell and/or the at least one additional target cell may be any cell of interest, for example it may be a cancer cell. It may be a circulating tumour cell. It may be a Hepatocellular carcinoma cell. It may be a cell expressing specific antigens, for example GPC3 antigen and EpCAM antigen. It may be a Hep3B cell. The method is suitable for detecting antigens at a low concentration, and therefore also cells at a low concentration. Having said this, the method is not limited to detection of such cells or antigens. Any antibody may be used, and therefore it may be applied widely to any antigen or cell type. Contacting the sample with the electrode may comprise incubating the electrode with the sample for at least 25 minutes. The typical incubation period for antigen or cell capture onto the electrode is from about 25 minutes to about 60 minutes. This is to allow for effective cell capture from the sample, while avoiding antibody and antigen/cell inactivation from overexposure to heat. The specific conditions may differ according to the choice of antibody and antigen/cell. For example, to allow capture of Hep3B cells via Anti-EpCAM attached to the electrode, the electrode was incubated for various time periods with a cell suspension having 1 *10⁶ cells mL^"1 and it was found that maximal capture occurred after 30 minutes at 37 degrees centigrade (see Fig. 9A).

Contacting the electrode with the first nanoparticle composition and second nanoparticle composition may comprise incubating the electrode with the first nanoparticle composition and second nanoparticle composition for at least 50 minutes. This is to allow for effective binding of the nanobioprobes (nanoparticles with antibodies and quantum dots) to the captured cells on the electrode, while avoiding antibody and antigen/cell inactivation from overexposure to heat. The specific conditions may differ according to the choice of antibody and antigen/cell. For example, to allow binding of nanoparticles to Hep3B cells via Anti-EpCAM and Anti-GPC3 attached to different nanoparticles, the electrode was incubated for various time periods with the different nanoparticles and it was found that maximal capture occurred after 50 minutes at 37 degrees centigrade (see Fig. 9B).

The third antibody may be the same as the first antibody or the second antibody. This is the case where the third antibody attached to the electrode for cell capture is the same antibody used in one of the first or second nanoparticles. This may provide ease of preparation of the apparatus. It may also aid in selecting for substances in the sample which have a high probability of being the substance of interest, e.g. the antigen of interest or the target cell of interest. Selecting for these substances may concentrate the binding of the nanoparticles via their respective attached antibodies to a fewer number of captured cells and allow for a stronger signal for each bound antigen or target cell.

The method may further comprise:

providing one or more additional nanoparticle compositions, each additional nanoparticle composition comprising an additional nanoparticle, an additional plurality of quantum dots and an additional antibody, where the additional antibody is capable of binding to the at least one antigen and where the additional plurality of quantum dots and the additional antibody are attached to the additional nanoparticle; and

contacting the electrode with the one or more additional nanoparticle compositions, wherein:

subsequently detecting the presence of the at least one antigen in the sample further comprises detecting at least one property of each of the additional pluralities of quantum dots bound to the electrode by way of the at least one antigen, at least one third antibody and at least one first or second antibody;

each of the first, second and additional pluralities of quantum dots are different; and each of the first, second and additional antibodies are different.

This method allows for more than two nanoparticle compositions, each with a plurality of a different quantum dot and a different antibody, so that more complex detection can be performed. For example, for detection of and differentiation between two different target cell types in a sample, where each target cell type has two specific antigens, the method could use four nanoparticle compositions each with a plurality of a first, second, third or fourth quantum dot and each having an antibody specific to one of the four antigens. The method could also use an electrode having one or more capture antibodies that can capture the two target cell types. Detecting for the four nanoparticles (e.g. by SWV analysis of the dissolved metal ions from the quantum dots thereon) could then provide information on the four antigens, and therefore the presence and concentration of the two target cell types. This could be useful for example to differentiate between a liver cancer cell and a breast cancer cell. In a particular example:

(i) the at least one antigen comprises a GPC3 antigen and an EpCAM antigen;

(ii) the first antibody is capable of binding to the GF^*C3 antigen and the second antibody is capable of binding to the EpCAM antigen;

(iii) the graphene oxide coated electrode comprises an interface material attached to the plurality of the third antibody, the third antibody being capable of binding to the

EpCAM antigen;

(iv) contacting the sample with the electrode comprises incubating the electrode with the sample for at least 30 minutes at 37 degrees centigrade;

(v) contacting the electrode with the first nanoparticle composition and second nanoparticle composition comprises incubating the electrode with the first nanoparticle composition and second nanoparticle composition for at least 50 minutes at 37 degrees centigrade; and

(vi) detecting at least one property of each of the first and second pluralities of quantum dots comprises dissolving the quantum dots to obtain metal ions and performing Square wave stripping voltammetric analysis on the metal ions to determine the identity and/or amount of the first and second pluralities of quantum dots.

In this example, the presence of GPC3 and EpCAM antigens in the sample is indicative of the presence of a Hep3B cell or part thereof in the sample. This method is capable of detecting 10 cells or fewer per mL of sample, as shown in the Figures 3 and 4.

Further, there is provided an apparatus for use in the method of the invention, comprising a graphene oxide-coated electrode and a plurality of a third antibody capable of binding to at least one antigen, the plurality of the third antibody being attached to the electrode and the graphene oxide coating on the electrode having been electrochemically reduced. Such an apparatus has the advantage of a long shelf life compared to available immunoassays.

The apparatus may comprise an electrode comprising a Gold electrode. Alternatively, the apparatus may comprise an electrode comprising a Glassy Carbon Electrode.

The apparatus may comprise an electrode comprising an interface material attached to the plurality of the third antibody. The interface material may comprise chitosan. The major function of chitosan is to immobilize GO on the electrode surface without reducing the electron transfer capability of GO. However both chitosan and GO do not have natural affinity to the antibodies to be coated. Therefore glutaraldehyde may be used as a crosslink reagent for attaching antibodies to the GO-chitosan complex. Glutaraldehyde is a five- carbon molecule with aldehyde groups at each end, which can react with the amino groups of the chitosan and antibody respectively, thereby providing a strong link between the antibody and the chitosan-GO complex. The chitosan may be attached to the plurality of the third antibody by reaction with glutaraldehyde. Alternatively, other suitable interface materials may be used. Other linkers may also be used to attach antibodies to the chitosan-GO complex and thus the electrode.

Further, there is provided a kit of parts for use in the method of the invention, comprising: the apparatus of the invention; a first nanoparticle composition, comprising a first nanoparticle, a first plurality of quantum dots and a first antibody, where the first antibody is capable of binding to at least one antigen and where the first plurality of quantum dots and the first antibody are attached to the first nanoparticle; and

The kit of parts may comprise one or more additional nanoparticle compositions, each additional nanoparticle composition comprising an additional nanoparticle, an additional plurality of quantum dots and an additional antibody, where the additional antibody is capable of binding to the at least one antigen and where the additional plurality of quantum dots and the additional antibody are attached to the additional nanoparticle, wherein

Examples

Reagents. Anti-EpCAM antibody and anti-GPC3 were obtained from Meridian Life Science Inc. (TN, USA). 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), (3-aminopropyl)-triethoxysilane (APTS), Acetic acid (HAc), Sodium acetate (NaAc), Bismuth nitrate pentahydrate and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (MO, USA). All other chemicals were of analytical grade and were used as received. Phosphate buffer solution (PBS) was prepared by mixing NaH₂P0₄ and Na₂HP0₄. Twice-distilled water was used throughout the study.

Apparatus. Square-wave-voltammetric (SWV) stripping measurements were performed with a CHI 660C electrochemical workstation (CH Instrument Co., Shanghai, China). A conventional three-electrode system consisting of a modified bismuth film modified glassy carbon electrode (BFE), a platinum wire, and a saturated calomel electrode (SCE) were used as the working, auxiliary, and reference electrodes in the electrochemical measurements, respectively. The morphology of the bare silica nanoparticles, CdTe QDs coated silica nanoparticles (Si/Cd), and ZnSe QDs coated silica (Si/Zn) nanoparticles were analyzed with a transmission electron microscope (TEM, S-2400N, HITACHI, Japan).

X-ray photoelectron spectroscopy (XPS) was performed with ESCALAB 250 Multitechnique Surface Analysis System (Thermo Electron Co., USA).

Phase-contrast and fluorescent images were acquired by a Nikon inverted microscopy (ECLIPSE TE-2000U, Nikon, Japan) equipped with a video camera (DS-U1 , Nikon, Japan).

Cell Culture. Hep3B, MCF-7, PP5 cells were cultured in a flask in RPMI 1640 medium (GIBCO) supplemented with 10% fetal calf serum (FCS, Sigma), penicillin (100 pg mL^"1), and streptomycin (100 g mL^"1) at 37 in a humidified atmosphere containing 5% C0₂. The cells in the exponential growth stage were collected and separated from the medium by centrifugation at 000 rpm for 5 min, and then washed thrice with sterile 0.01 M pH 7.4 PBS. The cell pellet was re-suspended in 10 mM pH 7.4 PBS to obtain a homogeneous cell suspension. Cell number was determined using a Petroff-Hausser cell counter (USA). Preparation of the Antibody (Ab)-Coupled Nanobioprobe. (a) Preparation of Si0₂ nanoparticles, CdTe and ZnSe. Synthesis of monodispersed Si0₂ nanoparticles was carried out according to the reported seed-growth methods (Chang et al., J. Colloid Interface Sci. 2005, 286, 536-542; Lei et al., Microporous Mesoporous Mater. 2006, 96, 127-134). CdTel (λ=550 nm), CdTe2 (λ=620 nm) and ZnSe were synthesized according to a previously reported procedure (Qian et al., Small, 2006, 2, 747-751; Zhang et al., J. Am. Chem. Soc. 2006, 128, 10171-10180; Qian et al., J. Phys. Chem. B 2006, 110, 9034-9040; Shavel et al., J. Phys. Chem. B 2004, 108, 5905-5908).^31"34 The solution with the QDs was further purified by ultrafiltration (Zhelev et al., Anal. Chem. 2006, 78, 321-330).

(b) Preparation of QDs coated silica nanoparticles. For preparation of CdTel QDs-coated silica nanoparticles (Si/Cd1 ), CdTe2 QDs-coated silica nanoparticles (Si/Cd2) and ZnSe- coated silica (Si/Zn) nanoparticles, 0.02 g silica nanospheres were first dispersed in 2 mL ethanol and treated with 0.4 mL APTS. After stirring for 6 h, the suspension was centrifuged, washed with ethanol and the washing repeated four times. After that, the amino-functionalized silica nanoparticles were harvested. They were then redispersed in a mixture of 2 mL CdTel QDs, CdTe2 QDs or ZnSe QDs and 1 mL EDC (20 mg mL^'1). The suspension was stirred at 4^°C for 12h. Unbound QDs were removed by successive centrifugation and washed several times with water. Finally, the as-prepared Si/Cd1 , Si/Cd2 and Si/Zn nanoparticles obtained were dispersed in water to a final volume of 1 mL. (c) Preparation of Ab-Coupled Nanobioprobe. To generate QDs-coated silica nanosphere immunological labels, 1 mL Si/Cd1 suspension was mixed with 1 mL anti-EpCAM (25 pg mL^"1 in 0.01 M pH 7.4 PBS); 1 mL Si/Cd2 suspension was mixed with 1 mL anti-GPC3 solution (25 pg mL^"1 in 0.01 M pH 7.4 PBS) and 1 mL Si/Zn suspension was mixed with 1 mL anti-GPC3 solution (25 pg mL^"1 in 0.01 M pH 7.4 PBS). Subsequently, 100 pL of freshly prepared EDC (20 mg mL^"1 in 0.01 M pH 7.4 PBS) and 100 pL of NHS (10 mg mL^"1 in 0.01 M pH 7.4 PBS) were added. After incubation at room temperature for 2h, free antibodies were removed by centrifugation and the pellet was washed with 0.01 M PBS for several times to yield the antibody-modified-Si/Cd1 nanoparticles (Si/Cd1/anti-EpCAM), Si/Cd2 nanoparticles (Si/Cd2/anti-GPC3) or Si/Zn nanoparticles (Si/Zn/anti-GPC3). Finally, the Si/Cd1/anti-EpCAM, Si/Cd2/anti-GPC3 and Si/Zn/anti-GPC3 nanoparticles were resuspended individually in 5 mL of 1 % BSA solution for 2h to block the excess amino- group and nonspecific binding sites of the immunological labels. After being centrifuged and washed with PBS, the resultant Si/Cd1/anti-EpCAM, Si/Cd2/anti-GPC3 and Si/Zn/anti- GPC3 nanoparticles were resuspended individually with 0.01 M of pH 7.4 PBS to a final volume of 5 mL and stored at 4^°C for later use. The fabrication of the immunological labels was illustrated in Scheme 1A. For control experiments, Cd/anti-EpCA or Zn/anti-GPC3 were prepared by incubating the mixture of 1 mL CdTe QDs solution and 200 pL of anti- EpCAM or 1 mL ZnSe QDs solution and 200 pL of anti-GPC3 solution in the presence of 300 pL 2 mg/mL EDC solution for 2h at room temperature. The free nonconjugated QDs were removed by ultrafiltration using a 50,000 MW filter. Fabrication of biosensors for SWV analysis, (a) Preparation of antibodies modified GCE electrode. GO (graphene oxide) was synthesized from graphite by the modified Hummers method (Li et a/., J. Am. Chem. Soc. 2009, 131, 5851-5857 and Liu er a/., J. Am. Chem. Soc. 2010, 132, 7279-7281 ). The as-synthesized graphite oxide was suspended in water and subjected to dialysis for one week to remove residual salts. After dried at 50^°C overnight, as-purified graphite oxide was exfoliated into GO by ultrasonicating a 0.05 wt% aqueous dispersion for 30 min. Unexfoliated graphite oxide was removed by a 5 min ultrafiltration at 2000 rcf. The glassy carbon electrode (GCE) with 3 mm diameter was polished to a mirror using 1.0, 0.3, and 0.05 pm alumina slurry followed by rinsing thoroughly with deionized water. After successive sonication in 1 :1 nitric acid, acetone, and deionized water, the electrode was rinsed with water and allowed to dry at room temperature. 5 pL of 0.5 mg mL^"1 GO solution was dropped on the pretreated GCE, which was dried in air. Then 3 μΙ_ of 0.05% chitosan solution was dropped on the GO film and dried in air. After electrochemical reduction of GO at -1.0 V in pH 7.4 PBS, the modified electrode was washed with water and incubated with 5 pL of 2.5% glutaraldehyde (in 50 mM PBS, pH 7.4) for 2h, followed by washing with water. 5 μΙ_ of 0.2 mg mL^"1 anti-EpCAM was then dropped onto the surface and incubated sequentially at room temperature for 60 min at 4°C overnight in a 100% moisture-saturated environment. Subsequently, excess antibody was removed with washing buffer. Finally, 5 μΙ_ of BSA blocking solution was dropped on the electrode surface and incubated for 60min at room temperature to block possible remaining active sites against nonspecific adsorption. The anti-EpCAM /chitosan/electrochemically reduced GO/ GCE electrodes were thus obtained. The anti- EpCAM/chitosan/GCE was similarly prepared and used for a comparative experiment. (b) Sandwich immunoassay with Ab-Coupled Nanobioprobe. A sandwich immunoassay using Si/Cd1/anti-EpCAM and Si/Zn/anti-GPC3 as label was shown in Scheme 1B. The anti-EpCAM modified GCE electrode was incubated in 1 ml_ Hep3B cell suspension at 37°C for 30 min to capture cells with the first immunoreaction. After being washed thoroughly with PBS, the cells bound-GCE electrode was exposed to 0.4 mL Si/Cd1/anti- EpCAM and Si/Zn/anti-GPC3 suspension at 37°C for 50 min to introduce the labels onto the GCE electrode through the second immunoreaction. After that, it was thoroughly rinsed with PBS containing 0.05% Tween to remove immunological labels due to the physical adsorption to complete the sandwich immunoreactions.

SWV analysis, (a) Fabrication of bismuth film modified glassy carbon electrode (BFE). The pretreated GCE electrode was immersed in a bismuth nitrate solution in acetate with a final pH of 2.0 and Bi(lll) ion concentration of 1.25 mg mL^"1 and applied potential of -1.2V (vs. SCE) for 120s.

(b) SWV analysis. The QDs remaining on the surface of cell were dissolved by 30 μΙ_ HN0₃ solution (0.1 mol/L). The solution containing the dissolved metal ions was then transferred into 3 mL acetate buffer (0.1 mol/L) at pH 4.5. The amount and identity of the dissolved metal ions were determined by stripping voltammetry. The analytical procedure involved 120s electrodeposition at -1.2V for three times (with 10s stirring between each accumulation period). After that, it was stripped by scanning from -1.2 to -0.6V using SWV measurements with 4mV potential steps, 25Hz frequency and 25mV amplitude.

Fluorescence analysis. The anti-EpCAM modified GCE electrode was incubated in 1 mL Hep3B solution at 37°C for 30 min to capture the cells. After being washed thoroughly with PBS, the cells bound GCE electrode was exposed to 0.4 mL Si/Cd1/anti-EpCAM and Si/Cd2/anti-GPC3 suspension at 37°C for 30 min to introduce the labels onto the GCE electrode surface. After that, it was thoroughly rinsed with PBS containing 0.05% Tween to remove immunological labels due to physical adsorption and observed under fluorescent microscope.

RESULTS AND DISCUSSION

Preparation of the Ab-Coupled Nanobioprobe. Silica nanoparticles with good monodispersion and similar surface morphology were synthesized according to the previously reported seed-growth method (Wu et a/., Anal. Chem. 2009, 81, 1600-1607). TEM images showed that the as-prepared silica nanoparticles had a chemically clean and homogenized structure, with a diameter of 100 + 3.0 nm (Figure 7A). Coating of QDs onto the surface of silica nanospheres was achieved through acylamide binding in the presence of EDC as the activator. In this case, APTS was first coupled to the hydroxyl group on the surface of silica nanospheres to yield an amino-terminated self-assembled monolayer. Subsequently, the carboxylic groups located on the surface of CdTe or ZnSe QDs reacted with amino distal points, which formed QDs-coated silica nanoparticles. The coating of CdTe (Figure 7B) or ZnSe (Figure 7C) QDs on silica nanospheres was confirmed by TEM images, demonstrating the deposition of QDs on the surface of silica nanospheres with a uniform distribution. The other carboxyl groups located on QDs surface can be further coupled to the amino groups of antibodies through acrylamide-bonding in the presence of EDC and NHS as activating reagents. Figure 8 showed the XPS spectra of the QDs coated silica nanoparticles before and after antibody coating. The XPS spectrum of the CdTe QDs functionalized silica nanoparticles exhibits the binding energy of the electrons for Cd3d at 405.3 and 412.1 eV (curve a, Figure 8A). The XPS spectrum of the ZnSe QDs functionalized silica nanoparticles exhibits the binding energy of the electrons for Zn2p at 1022 and 1048 eV (curve a, Figure 8B). A weaker N 1s line was exhibited at 400.0 eV for N-H group in the APTS (curve a, Figure 8A and Figure 8C). This confirmed that CdTe QDs or ZnSe QDs have been successfully coated on the surface of silica nanospheres. After further coupling with the antibodies, the. XPS spectra showed two decreased Cd3d (curve b, Figure 8A) or Zn2p (curve b, Figure 8B) and one enhanced N1s line (curve b, Figure 8A and Figure 8C) at the same binding energy with QDs-coated silica nanospheres. This was due to the coating of the antibody on the surface of QDs.

Characterization of the immunosensor. Graphene, especially electrochemically reduced GO, exhibits high conductivity and mediate electron transfer at its edge planes. It has been used in biosensors to improve detection sensitivity. The present invention employed reduced GO to construct a sensitive immunosensor for cell detection. After GO was doped in chitosan, the resulting chitosan/GO/GCE showed much lower electron transfer resistance, Ret (Figure 2(A), curves a and b). Upon the electrochemical reduction of GO, the Ret further decreased (Figure 2(A), curve c). The antibodies modified GCE was used to capture Hep3B cells from the cell suspension through the first immunoreaction. Due to the presence of EpCAM and GPC3 on the cell surface, Si/Cd/anti-EpCAM and Si/Zn/anti- GPC3 labels were introduced onto the cell surface through the secondary binding event. After the captured CdTe and ZnSe dissolved to generate Cd²⁺ and Zn²⁺ from GCE electrode in a 0.1 M HN0₃ solution, the captured CdTe and ZnSe QDs on GCE electrode could be detected by a BFE. Figure 2(B) shows the SWV curves of different electrodes with Hep3B cell of 1 *10⁶ cells ml^"1. A well-defined peak for the oxidation of Cd and Zn was observed from the immunosensor at around -0.75 V and -1.15 V (Figure 2(B), curves a and b). However, the oxidation current of 14.76 μΑ by Si/Cd/anti-EpCAM and 8.88 μΑ by Si/Zn/anti-GPC3 labels (Figure 2(B), curve a) was 2 times and 2.7 times larger than the 7.11 μΑ by Cd/anti-EpCAM and 3.26 μΑ by Zn/anti-GPC3 labels (Figure 2(B), curve b), respectively, showing signal amplification by silica nanoparticles. The signal amplification was due to the increase of the CdTe or ZnSe QDs loading in per immunological events. When graphene was not used, a lower oxidation current was obtained (Figure 2B, curve c), indicating that the presence of graphene greatly increased the density of anti-EpCAM on the surface of the nanobioprobe to further amplify the detection signal (Figure 2B, curve a).

To verify the signal amplification of graphene oxide, anti-EpCAM was directly coupled to the GCE through chitosan. Although anti-EpCAM/chitosan/GCE could also specifically capture the Hep3B cells, a comparatively lower oxidation current was obtained, (Figure 2(B), curve c) indicating that the presence of graphene greatly increased the density of anti-EpCAM on the biosensor surface and further enhanced the detection signal (Figure 2(B), curve a).

A series of control experiments were conducted. No peaks were observed after incubation of the antibodies modified GCE in a cell-free solution, followed by incubation in Si/Cd/anti- EpCAM and Si/Zn/anti-GPC3 suspension (Figure 2(B), curve d); no peaks were observed after incubation of the antibodies modified GCE in a solution of PP5 cells (EpCAM and GPC3 negative) solution, followed by incubation in Si/Cd/anti-EpCAM and Si/Zn/anti-GPC3 suspension (Figure 2(B), curve e); just one peak for Cd was observed after incubation of the antibodies modified GCE in a solution of MCF-7 cells (EpCAM positive, GPC3 negative), followed by incubation in Si/Cd/anti-EpCAM and Si/Zn/anti-GPC3 suspension (Figure 2(B), curve f). These observations confirmed that the SWV responses were attributed to the binding of QDs-coated silica nanoparticles through the sandwiched immunoreactions. When two peaks appeared simultaneously, it confirmed the presence of Hep3B cells (EpCAM and GPC3 positive). Additionally, the wide flat baseline (e.g., between the Cd and Zn peaks or before Cd) indicates the possibility of measuring additional protein biomarkers simultaneously on the cell surface.

Optimization of the Conditions for Immunoassay. Kinetically, the number of cells captured from the solution depends on the incubation time before it reaches thermodynamic equilibrium. By incubating antibodies modified GCE in the cell-containing solution for different period of time, the oxidation current of Cd was observed to reach a maximum value at 30 min, so the incubation time was chosen to be 30 min (Figure 9A). The amount of Si/Cd/anti-EpCAM and Si/Zn/anti-GPC3 labels immobilized on the cell surface was associated with the incubation time between labels and cells. With increasing incubation time, the oxidation current of Cd increased and trended to a constant value after an incubation time of 50 min (Figure 9B), which showed a saturated binding between the labels and the cells. Therefore, the incubation time of 50 min was selected for label immobilization. SWV Analytical Performance. Under optimum conditions, the stripping peak current of CdTe and ZnSe QDs immobilized on the immunosensor increased with increasing concentration of Hep3B cells in the incubation solution. The current of Cd was found to be proportional to the concentration of Hep3B cells in the incubation solution, which was within the calibration range from 5 to 1 x10⁶ cells mL^"1 (Figure 3). The linear curve fits a regression equation of log (/ρ/μΑ) = 0.29 - 0.59 log (C_ceii/cells mL^"1), where Ip is the oxidation current, and C is the cell concentration (Inset in Figure 3(B)). On repetition, results obtained were found to fit a regression equation of log (/ρ/μΑ) = 0.31 - 0.67 log (C_cen/cells mL^"1). The lowest detectable concentration was 5 cells mL^"1, which was much lower than those of 620 cells mL^"1 by an electrochemical cytosensor for detection of BGC cells⁴² and 800 cells mL^"1 by an electrochemiluminescent cytosensor for detection of HepG2 cells.⁴³ The current of the Zn was proportional to the concentration of Hep3B cells from 10 to 1 *10⁶ cells mL^"1 (Figure 4). The linear curve fits a regression equation of log (/ρ/μΑ) = 0.28 - 0.8 log (C_Ceii/cells mL^"1), the lowest detectable concentration was 10 cells mL^"1 (Inset in Figure 4(B)). On repetition, results obtained were found to fit a regression equation of log (/ρ/μΑ) = 0.29 - 0.83 log (C_ceii/cells mL^"1). These results demonstrate that the present method is highly sensitive, especially for the detection of CTCs at low concentration levels. Fluorescent Performance. Figure 5 shows the fluorescence microscope images of Ab- coupled nanobioprobes. Both the Si/Cd1/anti-EpCAM (Figure 5(A)) and Si/Cd2/anti-GPC3 (Figure 5(B)) were clearly monodispersed and retained the expected fluorescence. To confirm the Ab-coupled nanobioprobes maintaining the capability of recognizing the target cells, the Si/Cd1/anti-EpCAM and Si/Cd2/anti-GPC3 were incubated with Hep3B cells- coated GCE as mentioned in the Experimental Section. Because the target cancer cells had been stained with fluorescent dyes and the labels contained fluorescent quantum dots, they could be visualized by their different fluorescence when excited under a lamp-house after the experiment (Figure 5, panels C-E). When the blue fluorescence of nucleus and the red and green fluorescence representing the Si/Cd1/anti-EpCAM and Si/Cd2/aiiti-GPC3 nanobioprobes, respectively, appeared simultaneously on the cell surface of the tumor cells (Figure 5F) the positive identification of Hep3B liver cancer cells would be indicated. As controls for specificity, MCF-7 cells (EpCAM-positive, GPC3-negative) and PP5 cells (EpCAM and GPC3-negative) were also tested. For MCF-7 cells, the blue fluorescence of the nuclei and the red fluorescence of Si/Cd1/anti-EpCAM nanobioprobes could be detected (Figure 6, panels A and B, respectively). When these images were overlaid, the blue and the red fluorescence appeared simultaneously on the cell surface of MCF-7 cells (Figure 6C), demonstrating the presence of EpCAM antigen and the absence of GPC3 antigen on its cell surface. In comparison, no fluorescent signal could be detected for PP5 cells, demonstrating the absence of EpCAM and GPC3 antigens on its cell surface (Figure 6D).

Reproducibility, Precision, Stability and Regeneration of Immunosensor. Both the intra-assay and inter-assay precisions of the immunosensor were examined with 1 *10⁶ Hep3B cells for five times. The relative standard deviations (RSD) were 4.6% and 6.2%, respectively, showing good precision and acceptable fabrication reproducibility. In addition, when the immunosensor was stored in dry conditions at 4°C, over 90% of the initial response remained after a storage period of 2 weeks. These results indicated that the immunosensor had acceptable reliability and stability.

The regeneration step was performed by immersion of the working electrode for 10 min in 0.1 M glycine-HCI (pH=2.2) to interrupt the antigen-antibody immunocomplex and could offer the potential to harvest the bound tumor cells for further molecular and biochemical characterizations. After each sandwich immunoassay, the electrode was immersed in 0.1 M pH 2.2 glycine-HCI for 10 min. No detectable SWV signals could be detected while incubation of this regenerated electrode in Hep3B cell suspension, followed by Si/Cd/anti- EpCAM and Si/Zn/anti-GPC3 suspension gave comparable SWV responses as earlier described. Relative standard deviation of 6.5% and 8.7% was obtained, respectively, for the Cd and Zn nanobioprobes, following 6 repeated detection-regeneration cycles for Cd and Zn, respectively. This observation also demonstrated that the as-synthesized Si/Cd/anti-EpCAM and Si/Zn/anti-GPC3 possesses good monodispersion characteristics to enable the consistent loading of the same amount of QDs on each microsphere. The apparent uniformity in dispersion provides an added advantage for its application in the clinical diagnosis of CTCs.

CONCLUSIONS

An ultrasensitive and highly specific electrochemical and fluorescent immunosensing method for the detection of circulating tumor cells was achieved using a dual signal amplification strategy based on nanobiotechnology. The introduction of graphene on the immunosensor surface efficiently accelerated the electron transfer and enhanced the detection signal. The second signal amplification came from the loading of QDs on silica nanoparticle surface. After immunoreactions, two kinds of tracing tags were co-immobilized on the cell surface that could be conveniently detected by anodic stripping analysis and fluorescence microscopy. In addition, the regeneration of the immunosensor enables the elution and harvesting of the captured CTCs cells for further molecular analysis. The present immunosensor as design described showed high sensitivity and specificity to detect low-abundance tumor cells with excellent stability, reproducibility and accuracy, indicating its wide potential applications for clinical diagnostics.

Claims

1. A method for detecting at least one antigen in a sample, comprising:

(iv) contacting the sample with the electrode;

the graphene oxide coating on the electrode has been electrochemically reduced.

2. The method according to claim 1 , wherein (iv) further comprises removing unbound sample from the electrode after contacting the sample with the electrode and/or (v) further comprises removing unbound nanoparticle compositions from the electrode after contacting the electrode with the first nanoparticle composition and the second nanoparticle composition.

3. The method according to claim 1 or claim 2, wherein detecting at least one property of each of the first and second pluralities of quantum dots comprises dissolving the first and second pluralities of quantum dots to obtain metal ions and performing voltammetric analysis on the metal ions to determine the identity and/or amount of the first and second pluralities of quantum dots.

4. The method according to claim 3, wherein the voltammetric analysis comprises Square wave stripping voltammetry.

5. The method according to any one of the preceding claims, wherein detecting at least one property of each of the first and second pluralities of quantum dots further comprises detecting the fluorescence of the first and second pluralities of quantum dots.

6. The method according to any one of the preceding claims, wherein the at least one antigen comprises at least two antigens specific to a target cell, wherein the presence ^ the at least two antigens is indicative of the presence of the target cell.

7. The method according to claim 6, wherein the at least one antigen comprises at least two additional antigens specific to at least one additional target cell, wherein the presence of the at least two additional antigens is indicative of the presence of the at least one additional target cell.

8. The method according to claim 7, further comprising a step of eluting the bound target cell from the electrode, optionally including a step of eluting bound nanoparticles from the target cell, and/or the method according to claim 6 further comprising a step of eluting the bound at least one additional target cell from the electrode, optionally including a step of eluting bound nanoparticles from the at least one additional target cell.

9. The method according to claim 8, wherein the target cell and/or the at least one additional target cell is a cancer cell.

10. The method according to claim 9, wherein the target cell and/or the at least one additional target cell is a circulating tumour cell.

11. The method according to claim 10, wherein the target cell and/or the at least one additional target cell is a Hepatocellular carcinoma cell.

12. The method according to claim 11 , wherein the target cell and/or the at least one additional target cell is a cell expressing GPC3 antigen and EpCAM antigen.

13. The method according to claim 12, wherein the target cell and/or the at least one additional target cell is a Hep3B cell.

14. The method according to any one of the preceding claims, wherein contacting the sample with the electrode comprises incubating the electrode with the sample for at least 25 minutes.

15. The method according to any one of the preceding claims, wherein contacting the electrode with the first nanoparticle composition and second nanoparticle composition comprises incubating the electrode with the first nanoparticle composition and second nanoparticle composition for at least 50 minutes.

16. The method according to any one of the preceding claims, wherein the third antibody is the same as the first antibody or the second antibody.

17. The method according to any one of the preceding claims, further comprising

18. The method according to any one of the preceding claims, wherein:

(i) the at least one antigen comprises a GPC3 antigen and an EpCAM antigen;

(ii) the first antibody is capable of binding to the GPC3 antigen and the second antibody is capable of binding to the EpCAM antigen;

(iii) the graphene oxide coated electrode comprises an interface material attached to the plurality of the third antibody, the third antibody being capable of binding to the EpCAM antigen; (iv) contacting the sample with the electrode comprises incubating the electrode with the sample for at least 30 minutes at 37 degrees centigrade;

19. The method according to claim 18, wherein the presence of GPC3 and EpCAM antigens in the sample is indicative of the presence of a Hep3B cell or part thereof in the sample.

20. An apparatus for use in the method of any one of claims 1 to 19, comprising a graphene oxide-coated electrode and a plurality of a third antibody capable of binding to at least one antigen, the plurality of the third antibody being attached to the electrode and the graphene oxide coating on the electrode having been electrochemically reduced.

21. The apparatus according to claim 20, wherein the electrode comprises a Gold Electrode.

22. The apparatus according to claim 20, wherein the electrode comprises a Glassy Carbon Electrode.

23. The apparatus according to any one of claims 20 to 22, wherein the electrode comprises an interface material attached to the plurality of the third antibody.

24. The apparatus according to claim 23, wherein the interface material comprises chitosan.

25. The apparatus according to claim 24, wherein the chitosan is attached to the plurality of the third antibody by reaction with glutaraldehyde.

26. A kit of parts for use in the method of any one of claims 1 to 19, comprising: the apparatus of any one of claims 20 to 25;

27. The kit of parts according to claim 26, comprising one or more additional nanoparticle compositions, each additional nanoparticle composition comprising an additional nanoparticle, an additional plurality of quantum dots and an additional antibody, where the additional antibody is capable of binding to the at least one antigen and where the additional plurality of quantum dots and the additional antibody are attached to the additional nanoparticle, wherein