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An AC electrokinetic impedance immunosensor for rapid detection of tuberculosis Haochen Cui,a Shanshan Li,ab Quan Yuan,a Ashutosh Wadhwa,c Shigetoshi Eda,c Mark Chambers,d Roland Ashford,d Hongyuan Jiangb and Jie Wu*a This work presents an AC electrokinetic impedance sensing method that is capable of detecting specific interactions between macromolecules such as antigen–antibody binding. Serum samples were added to the surface of interdigitated electrodes that had been coated with bacterial antigens. After applying an AC signal of 100 mV at a specific frequency continuously, the electrodes' impedance change was recorded and used to determine the occurrence and level of antibody binding to the antigen. Our theoretical analysis indicated that with this AC signal, the target macromolecules will experience a sufficiently strong attraction force towards the electrode surface for acceleration of the binding process.
Received 4th June 2013 Accepted 7th October 2013
Using this method, 11 human tuberculosis and 10 bovine tuberculosis serum samples were tested. The results were consistent with those obtained by a conventional ELISA method. The limit of detection of the impedance sensing method was estimated to be better than 10 ng mL1. In summary, we demonstrate that AC electrokinetic impedance sensing can be used for rapid and sensitive detection of
DOI: 10.1039/c3an01112g
specific antibodies in serum samples. This method may form a basis for development of a point of care
www.rsc.org/analyst
diagnostic device for human and bovine tuberculosis.
1.
Introduction
Tuberculosis (TB) is a chronic infectious disease that affects a broad range of mammalian hosts including humans, cattle, deer, llamas, wild boar, domestic cats, wild carnivores (fox, coyotes, badgers), possums, and rodents. Human TB, caused by Mycobacterium tuberculosis, occurs in more than ten million people worldwide, and is estimated to be responsible for the death of two million people annually. It is also estimated that over one billion dollars is spent on diagnosis and evaluation of human TB worldwide each year.1 Although bovine TB, caused by Mycobacterium bovis, has been mostly eradicated in the livestock industry of developed countries, the disease in wildlife still poses a risk to livestock, tourism economy, and wildlife conservation. Global economic losses from bovine TB total US$ 3 billion annually. In the United States, $40 million (and in Great Britain, £100 million) were spent on bovine TB management in the year 2008–2009 alone. In developing countries, bovine TB still causes serious concerns not only for wildlife, but
a
Department of Electrical Engineering and Computer Science, The University of Tennessee, Knoxville, TN 37996, USA. E-mail: [email protected]
b
School of Mechatronics Engineering, Harbin Institute of Technology, 150001, PR China
c Department of Forestry, Wildlife and Fisheries, The University of Tennessee, Knoxville, TN 37996, USA d
Department of Bovine Tuberculosis, Animal Health and Veterinary Laboratories Agency, Addlestone, Surrey, KT15 3NB, UK
7188 | Analyst, 2013, 138, 7188–7196
also for public health, food safety and the economy of livestock industries. Conventional methods of TB diagnosis are labor-intensive, time-consuming and/or expensive. For example, medical evaluation of human TB includes a medical history, physical examination, radiographic imaging (conventional chest X-ray), microbiological smears, and bacterial cultures. It may also include a tuberculin skin test or other measurements of cellular immunity, such as interferon-gamma release assays (IGRA). For human TB, the Cepheid Xpert MTB/RIF assay has recently been used for on-site diagnosis but the assay is costly.2,3 Bovine TB in animals is commonly conrmed by post mortem examination of gross lesions and bacterial culture. Animals can also be diagnosed before death by immunodiagnosis (including intradermal tuberculin skin testing and IGRA) and culture of clinical samples. Global control and prevention of TB has partly been hampered by the lack of effective diagnostic methods, so there is an urgent need for more simple, rapid and sensitive on-site diagnosis of TB.4 In recent years, immunosensors have attracted a great amount of research effort because of their potential to achieve rapid, inexpensive, and portable on site diagnosis.5 Immunosensors can detect specic analytes of interest from a biological solution through the use of a functionalized sensor surface, i.e. by immobilizing a capture probe on the sensor surface prior to adding a biological sample solution. The most studied immunosensors include induced uorescence immunoassay,6 giant magnetoresistive sensors,7 surface plasmon resonance8 and
This journal is ª The Royal Society of Chemistry 2013
View Article Online
Published on 07 October 2013. Downloaded by University of California - Santa Cruz on 22/10/2014 10:32:43.
Paper microelectrode impedimetric sensors.9,10 Fluorescent immunoassay is one of the most widely used methods; however, it requires multiple steps for antibody incubation and washing, making it too time-consuming to be used for point of care diagnosis. Giant magnetoresistive sensors have also been used to quantify immunological interactions at the sensor surface, however, similar to uorescent immunoassay, they require a labeling step with magnetic particles. Although surface plasmon resonance is a label-free sensing method, it requires sophisticated optical instrumentation and highly trained personnel to operate. Compared with the aforementioned immunosensors, microelectrode impedimetric immunosensors are rather advantageous due to their simplicity in operation and their low cost of fabrication. Currently, research on impedimetric immunosensors is directed in several directions,11 such as improving immobilization methods of receptors,12 exploring nanostructured electrodes to amplify the detection signal,9 and investigating equivalent circuit or data processing algorithms to effectively correlate analyte concentrations with impedance changes. While signicant progress has been made in electrical impedance sensors, for them to be used towards on-site detection, there are still some obstacles to be overcome. First, many impedimetric tests would require sophisticated and oen timeconsuming data processing to extract binding related information. Expensive benchtop impedance analyzers are usually needed if high test frequencies (>1 MHz) are used. Second, most of the reported impedance assays are conducted with highly processed samples. To be viable as an on-site diagnostic system, sample preparation must be simple enough to be performed on site. Third, most of the currently tested systems still require at least 30 minutes to perform a single assay on abundant molecules or longer time for more diluted analytes. While this is an improvement over the standard enzyme-linked immunosorbent assay (ELISA) method, an incubation time of half an hour or more may still be too long under some circumstances. For example, testing multiple wild animals in eld would be much more feasible if a more rapid test becomes available. AC electrokinetic (ACEK) microuidics13 emerged in the 1990s and has been intensively studied as a means to manipulate particles or macromolecules. It has been demonstrated by several groups14–17 that ACEK working with microelectrodes can induce in situ concentration of particles for improved detection sensitivity and throughput. Our previous work has investigated ACEK concentrations of biomolecules by both numerical study18 and experiments.19,20 In this study, a new impedance assay was developed by incorporating the AC electrokinetic (ACEK) effect seamlessly with the signal interrogation process for in situ macromolecule enrichment and binding process monitoring. Unlike conventional impedimetric sensing, which uses a low AC voltage of 5–10 mV before and aer binding, our method reads the electrode impedance continuously with a voltage of 100 mVrms at a specic frequency. Such an AC signal would be suitable for inducing ACEK effects to convect/attract macromolecules towards the electrode surface. It has been demonstrated by our previous work that the binding time has been greatly reduced from hours to minutes, when compared with the conventional
This journal is ª The Royal Society of Chemistry 2013
Analyst method using a static uid chamber (hours). Our previous study found that changes in the level and frequency of AC potential will lead to corresponding changes in the magnitude of sensor response.19,21 Therefore, it is surmised that the shortened detection time in our technology is due to ACEK effects that exert attraction force on macromolecules towards the electrode surface, hence accelerating binding reactions. We also carefully choose our electrode design and detection protocol, so that at the AC enrichment frequency, the detection cell can be represented with a simplied resistor–capacitor serial connection (solution resistance in series with electrode/uid interfacial capacitance). Because the effect of sample matrix on cell impedance is mostly evident in its resistive component (e.g. charge transfer resistance), our detection method and device combined is relatively unsusceptible to interference from the background solution. As a result, our method needs little pretreatment of the sample (sample pre-treatment requires only volume dilution of serum samples with physiological buffer), and does not require a washing step aer binding, which is a major advantage over other immunosensors. It should be noted that the same AC signal is used to measure the impedance between microelectrodes, which indicates the binding activity and level occurring on the microelectrodes. So in the ACEK-based impedimetric assay, analyte enrichment and impedimetric detection are conducted simultaneously. Incorporation of ACEK with sensors has been investigated for various types of sensors, such as in microcantilevers22 uorescent labeled affinity sensing21 and impedance measurements.23–25 In all the aforementioned methods, the ACEK concentration step is separate from the detection step, which is more complicated to operate than the impedimetric assay reported in this work. In summary, the ACEK based impedimetric assay has the following merits, single step operation (it involves only applying the AC signal), label free, short “sample to results” time, reduced reagent cost, simple device design and reliable operation because of little human intervention during the test. This paper is organized as follows. Section 2 introduces the mechanism of ACEK impedance immunosensing. Devices and samples are described in Section 3, sensor characterization in Section 4, serum experimental results in Section 5, and limit of detection in Section 6. Finally, conclusions are made in Section 7.
2.
Mechanism
2.1
Impedance based immunosensing
Electrochemical impedance spectrum (EIS) analysis is a wellestablished method for characterizing an electrolytic cell. In EIS, a network of electronic components can be developed to represent an electrochemical cell.26 A change in the EIS spectra can be correlated with a change in the (di)electric properties of the sample solution or the electrode/electrolyte interface. Our impedance immunosensor utilizes an array of interdigitated microelectrodes, which is obtained by modifying commercially available electrode chips, i.e. surface acoustic wave (SAW) resonator chips (PARS 433.92, AVX Corp.). Images of
Analyst, 2013, 138, 7188–7196 | 7189
View Article Online
Published on 07 October 2013. Downloaded by University of California - Santa Cruz on 22/10/2014 10:32:43.
Analyst original and modied SAW chips are shown in Fig. 1a, and the scanning electron micrograph of Fig. 1b shows the interdigitated electrode array in the SAW chip. Before the tests, antigen specic to the analyte antibody is immobilized on the surface of electrodes. When an antibody-containing solution is loaded, the interaction between antibodies and antigens will cause a change in the impedance of the electrode/electrolyte system, which can be sensed by an external impedance readout system to realize the detection of the antibody–antigen interaction. The impedance between a pair of electrodes immersed in liquid can be approximated as a network of interfacial capacitors and resistive components including charge transfer resistance and electrolyte resistance, as conceptually shown in Fig. 1c. Impedance immunosensors usually exploit the change in thickness or dielectric properties of the dielectric layer at the electrolyte–dielectric interface, which will lead to a change in the (interfacial) capacitance of the device during antibody– antigen interaction. The interfacial capacitor consists of a series connection of equivalent capacitors caused by electrical double layer (EDL) and macromolecule deposition. When a solid material is immersed into an electrolytic solution, the solid surface will acquire surface charges. To maintain charge neutrality, a thin layer of counter ions is formed at the solid/liquid interface to neutralize the surface charges at the solid surface, which is commonly known as the EDL. Electrically, EDL can be modeled as a capacitor. The layers of counter ions and surface charges are equivalent to the two plates in a capacitor, and the plate separation distance is the EDL thickness. When macromolecules are adsorbed onto the electrode surface, the interfacial capacitance Cint will change due to the change in the thickness and surface area of Cint, which can then be used to indicate the deposition of macromolecules or particles on the electrode as well as to correlate with the macromolecule or particle concentration in the uid. As shown in Fig. 2, prior to the affinity assay, the electrode surface is functionalized with a layer of probe molecules (antigens in this work) to achieve specicity for the targeted antibody. The interfacial capacitance can be approximated as 3s Cint ¼ 3s Aint dag þ dedl ; (1) 3p where 3s and 3p are the permittivities of the sample solution and the probe protein (antigen). The relative permittivity of protein
Paper is around 2–3,27 while that of water is 80. Aint is the electrode area, dag is the antigen thickness, and dedl is the EDL thickness, approximated by the Debye length of the buffer solution according to the Debye–Huckel theory.28 During the assay, the sample solution containing the target antibody will be introduced. When the binding of antigen–antibody takes place, the interfacial capacitance Cint is expected to change to 3s 3s dab þ dag þ dedl ; (2) Cint;binding ¼ 3s Aint;binding 3t 3p where 3t is the permittivity of the target protein, and Aint,binding is the effective area of the interfacial capacitor. The change in Cint could be either an increase or a decrease, as conceptually shown in Fig. 2a and b. As a result of the binding, antibodies are deposited onto the surface. The thickness of the dielectric layer could increase, which could cause a decrease in the interfacial capacitance. On the other hand, randomly deposited antibodies could cause an increase in the capacitor's surface area due to extra topology introduced by the antibody, especially when the probe molecules (antigens) are spaced apart, leading to a higher interfacial capacitance, as shown in Fig. 2b. Both changes could occur during the binding. Oen, one type of change dominates over the other, and then the detection of binding is possible. The situation in Fig. 2a, i.e. a decrease in Cint due to antigen– antibody binding, was commonly observed.26 In a diluted buffer solution, the EDL is relatively thick. As EDL envelops the antibodies on the electrodes, ne features on the scale of EDL thickness (dedl) will be lost, and the Cint change will be dominated by an increase in its thickness, i.e. Cint reduces. When EDL thickness is comparable to that of antibody topology, a positive change of Cint is possible, especially in a buffer solution of high ionic strength.29 Our work found that both an increase and a decrease in Cint could possibly arise from antigen–antibody binding, since an increase of Cint was consistently observed in human TB detection and a decrease in bovine TB detection. Preliminary experiments were conducted to support the above hypothesis about the interfacial capacitance change. Buffer solutions of two different ionic strengths, 1 mM and 2 mM phosphate buffer solutions (PBS), were used. The results corroborate that the EDL thickness (nm) relative to the characteristic length of the deposited macromolecules will determine whether a decrease or an increase in interfacial capacitance would result from binding reactions.
Fig. 1 (a) Commercially available electrode chips, i.e. surface acoustic wave (SAW) resonator chips (PARS 433.92, AVX Corp.). The left one was modified for immunosensing. (b) A scanning electron micrograph of the microelectrode array in the SAW chip. (c) Equivalent circuit for a pair of microelectrodes immersed in an electrolyte. Cint: interfacial capacitance, Rct: charge transfer resistance, Rs: electrolyte resistance, Cs: electrolyte capacitance.
7190 | Analyst, 2013, 138, 7188–7196
This journal is ª The Royal Society of Chemistry 2013
View Article Online
Published on 07 October 2013. Downloaded by University of California - Santa Cruz on 22/10/2014 10:32:43.
Paper
Analyst
Fig. 2 Two possible topology changes at the solid/fluid interface due to the protein binding reaction. (a) The thickness of the interfacial layer increases while its surface area decreases, Cint reduces as a result; and (b) when the increase in the surface area of Cint dominates over the changes in its thickness, Cint increases (Ab: antibody; Ag: antigen).
2.2
ACEK acceleration of the binding reaction
For affinity based sensing, the detection of binding event usually relies on the diffusion of target macromolecules to the binding sites. It could take hours or even days for the amount of bound molecules to reach a detectable level.30 Therefore, great effort has been devoted to develop a bioparticle concentration mechanism to shorten the detection time and increase the sensitivity. ACEK, as a particle and uid manipulation mechanism, has minimal requirements on the device fabrication and operation to be incorporated into a detection system – only microelectrodes and their AC signal source need to be added. ACEK effects use an AC electric eld to induce particle and uid movement, so that macromolecules can be in situ concentrated onto microsensors.31 When an inhomogeneous AC electric eld is applied to an aqueous solution, both particle movement and microows can be induced to transport particles. Direct particle movement can be caused by dielectrophoresis (DEP), and particle can also be carried by microows such as AC electroosmosis or AC electrothermal ows to reach the microsensor. The manipulation of particles by DEP is based on the difference between the particle polarizability and that of the medium solution17 at a certain frequency. DEP force on a spherical particle can be expressed as follows: # 3*p 3*m 2 V |E| ; ¼ p3m a Re * 3p þ 23*m "
FDEP
3
(3)
where a is the radius of the particle, and 3*p and 3*m are particle and medium complex permittivities, respectively, given as 3* ¼ 3 + is/u (s: conductivity; u: angular frequency). The term in the bracket, known as the Clausius–Mossotti factor, strongly depends on AC frequency and buffer conditions, and determines the polarity of DEP force. When the Clausius–Mossotti factor is positive, the particle will experience a positive DEP force, moving towards high electric eld regions, such as electrode edges. When the Clausius–Mossotti factor is negative, the particles will experience a negative DEP force, being repelled from electrodes. Positive DEP is employed for particle concentration in this work.
This journal is ª The Royal Society of Chemistry 2013
As it can be seen from eqn (3), the magnitude of DEP force depends on the particle volume. In the case of nanoscale macromolecules, DEP may not be effective unless the molecules are located within a very short distance to the electrodes (
Published on 07 October 2013. Downloaded by University of California - Santa Cruz on 22/10/2014 10:32:43.
PAPER
Cite this: Analyst, 2013, 138, 7188
View Journal | View Issue
An AC electrokinetic impedance immunosensor for rapid detection of tuberculosis Haochen Cui,a Shanshan Li,ab Quan Yuan,a Ashutosh Wadhwa,c Shigetoshi Eda,c Mark Chambers,d Roland Ashford,d Hongyuan Jiangb and Jie Wu*a This work presents an AC electrokinetic impedance sensing method that is capable of detecting specific interactions between macromolecules such as antigen–antibody binding. Serum samples were added to the surface of interdigitated electrodes that had been coated with bacterial antigens. After applying an AC signal of 100 mV at a specific frequency continuously, the electrodes' impedance change was recorded and used to determine the occurrence and level of antibody binding to the antigen. Our theoretical analysis indicated that with this AC signal, the target macromolecules will experience a sufficiently strong attraction force towards the electrode surface for acceleration of the binding process.
Received 4th June 2013 Accepted 7th October 2013
Using this method, 11 human tuberculosis and 10 bovine tuberculosis serum samples were tested. The results were consistent with those obtained by a conventional ELISA method. The limit of detection of the impedance sensing method was estimated to be better than 10 ng mL1. In summary, we demonstrate that AC electrokinetic impedance sensing can be used for rapid and sensitive detection of
DOI: 10.1039/c3an01112g
specific antibodies in serum samples. This method may form a basis for development of a point of care
www.rsc.org/analyst
diagnostic device for human and bovine tuberculosis.
1.
Introduction
Tuberculosis (TB) is a chronic infectious disease that affects a broad range of mammalian hosts including humans, cattle, deer, llamas, wild boar, domestic cats, wild carnivores (fox, coyotes, badgers), possums, and rodents. Human TB, caused by Mycobacterium tuberculosis, occurs in more than ten million people worldwide, and is estimated to be responsible for the death of two million people annually. It is also estimated that over one billion dollars is spent on diagnosis and evaluation of human TB worldwide each year.1 Although bovine TB, caused by Mycobacterium bovis, has been mostly eradicated in the livestock industry of developed countries, the disease in wildlife still poses a risk to livestock, tourism economy, and wildlife conservation. Global economic losses from bovine TB total US$ 3 billion annually. In the United States, $40 million (and in Great Britain, £100 million) were spent on bovine TB management in the year 2008–2009 alone. In developing countries, bovine TB still causes serious concerns not only for wildlife, but
a
Department of Electrical Engineering and Computer Science, The University of Tennessee, Knoxville, TN 37996, USA. E-mail: [email protected]
b
School of Mechatronics Engineering, Harbin Institute of Technology, 150001, PR China
c Department of Forestry, Wildlife and Fisheries, The University of Tennessee, Knoxville, TN 37996, USA d
Department of Bovine Tuberculosis, Animal Health and Veterinary Laboratories Agency, Addlestone, Surrey, KT15 3NB, UK
7188 | Analyst, 2013, 138, 7188–7196
also for public health, food safety and the economy of livestock industries. Conventional methods of TB diagnosis are labor-intensive, time-consuming and/or expensive. For example, medical evaluation of human TB includes a medical history, physical examination, radiographic imaging (conventional chest X-ray), microbiological smears, and bacterial cultures. It may also include a tuberculin skin test or other measurements of cellular immunity, such as interferon-gamma release assays (IGRA). For human TB, the Cepheid Xpert MTB/RIF assay has recently been used for on-site diagnosis but the assay is costly.2,3 Bovine TB in animals is commonly conrmed by post mortem examination of gross lesions and bacterial culture. Animals can also be diagnosed before death by immunodiagnosis (including intradermal tuberculin skin testing and IGRA) and culture of clinical samples. Global control and prevention of TB has partly been hampered by the lack of effective diagnostic methods, so there is an urgent need for more simple, rapid and sensitive on-site diagnosis of TB.4 In recent years, immunosensors have attracted a great amount of research effort because of their potential to achieve rapid, inexpensive, and portable on site diagnosis.5 Immunosensors can detect specic analytes of interest from a biological solution through the use of a functionalized sensor surface, i.e. by immobilizing a capture probe on the sensor surface prior to adding a biological sample solution. The most studied immunosensors include induced uorescence immunoassay,6 giant magnetoresistive sensors,7 surface plasmon resonance8 and
This journal is ª The Royal Society of Chemistry 2013
View Article Online
Published on 07 October 2013. Downloaded by University of California - Santa Cruz on 22/10/2014 10:32:43.
Paper microelectrode impedimetric sensors.9,10 Fluorescent immunoassay is one of the most widely used methods; however, it requires multiple steps for antibody incubation and washing, making it too time-consuming to be used for point of care diagnosis. Giant magnetoresistive sensors have also been used to quantify immunological interactions at the sensor surface, however, similar to uorescent immunoassay, they require a labeling step with magnetic particles. Although surface plasmon resonance is a label-free sensing method, it requires sophisticated optical instrumentation and highly trained personnel to operate. Compared with the aforementioned immunosensors, microelectrode impedimetric immunosensors are rather advantageous due to their simplicity in operation and their low cost of fabrication. Currently, research on impedimetric immunosensors is directed in several directions,11 such as improving immobilization methods of receptors,12 exploring nanostructured electrodes to amplify the detection signal,9 and investigating equivalent circuit or data processing algorithms to effectively correlate analyte concentrations with impedance changes. While signicant progress has been made in electrical impedance sensors, for them to be used towards on-site detection, there are still some obstacles to be overcome. First, many impedimetric tests would require sophisticated and oen timeconsuming data processing to extract binding related information. Expensive benchtop impedance analyzers are usually needed if high test frequencies (>1 MHz) are used. Second, most of the reported impedance assays are conducted with highly processed samples. To be viable as an on-site diagnostic system, sample preparation must be simple enough to be performed on site. Third, most of the currently tested systems still require at least 30 minutes to perform a single assay on abundant molecules or longer time for more diluted analytes. While this is an improvement over the standard enzyme-linked immunosorbent assay (ELISA) method, an incubation time of half an hour or more may still be too long under some circumstances. For example, testing multiple wild animals in eld would be much more feasible if a more rapid test becomes available. AC electrokinetic (ACEK) microuidics13 emerged in the 1990s and has been intensively studied as a means to manipulate particles or macromolecules. It has been demonstrated by several groups14–17 that ACEK working with microelectrodes can induce in situ concentration of particles for improved detection sensitivity and throughput. Our previous work has investigated ACEK concentrations of biomolecules by both numerical study18 and experiments.19,20 In this study, a new impedance assay was developed by incorporating the AC electrokinetic (ACEK) effect seamlessly with the signal interrogation process for in situ macromolecule enrichment and binding process monitoring. Unlike conventional impedimetric sensing, which uses a low AC voltage of 5–10 mV before and aer binding, our method reads the electrode impedance continuously with a voltage of 100 mVrms at a specic frequency. Such an AC signal would be suitable for inducing ACEK effects to convect/attract macromolecules towards the electrode surface. It has been demonstrated by our previous work that the binding time has been greatly reduced from hours to minutes, when compared with the conventional
This journal is ª The Royal Society of Chemistry 2013
Analyst method using a static uid chamber (hours). Our previous study found that changes in the level and frequency of AC potential will lead to corresponding changes in the magnitude of sensor response.19,21 Therefore, it is surmised that the shortened detection time in our technology is due to ACEK effects that exert attraction force on macromolecules towards the electrode surface, hence accelerating binding reactions. We also carefully choose our electrode design and detection protocol, so that at the AC enrichment frequency, the detection cell can be represented with a simplied resistor–capacitor serial connection (solution resistance in series with electrode/uid interfacial capacitance). Because the effect of sample matrix on cell impedance is mostly evident in its resistive component (e.g. charge transfer resistance), our detection method and device combined is relatively unsusceptible to interference from the background solution. As a result, our method needs little pretreatment of the sample (sample pre-treatment requires only volume dilution of serum samples with physiological buffer), and does not require a washing step aer binding, which is a major advantage over other immunosensors. It should be noted that the same AC signal is used to measure the impedance between microelectrodes, which indicates the binding activity and level occurring on the microelectrodes. So in the ACEK-based impedimetric assay, analyte enrichment and impedimetric detection are conducted simultaneously. Incorporation of ACEK with sensors has been investigated for various types of sensors, such as in microcantilevers22 uorescent labeled affinity sensing21 and impedance measurements.23–25 In all the aforementioned methods, the ACEK concentration step is separate from the detection step, which is more complicated to operate than the impedimetric assay reported in this work. In summary, the ACEK based impedimetric assay has the following merits, single step operation (it involves only applying the AC signal), label free, short “sample to results” time, reduced reagent cost, simple device design and reliable operation because of little human intervention during the test. This paper is organized as follows. Section 2 introduces the mechanism of ACEK impedance immunosensing. Devices and samples are described in Section 3, sensor characterization in Section 4, serum experimental results in Section 5, and limit of detection in Section 6. Finally, conclusions are made in Section 7.
2.
Mechanism
2.1
Impedance based immunosensing
Electrochemical impedance spectrum (EIS) analysis is a wellestablished method for characterizing an electrolytic cell. In EIS, a network of electronic components can be developed to represent an electrochemical cell.26 A change in the EIS spectra can be correlated with a change in the (di)electric properties of the sample solution or the electrode/electrolyte interface. Our impedance immunosensor utilizes an array of interdigitated microelectrodes, which is obtained by modifying commercially available electrode chips, i.e. surface acoustic wave (SAW) resonator chips (PARS 433.92, AVX Corp.). Images of
Analyst, 2013, 138, 7188–7196 | 7189
View Article Online
Published on 07 October 2013. Downloaded by University of California - Santa Cruz on 22/10/2014 10:32:43.
Analyst original and modied SAW chips are shown in Fig. 1a, and the scanning electron micrograph of Fig. 1b shows the interdigitated electrode array in the SAW chip. Before the tests, antigen specic to the analyte antibody is immobilized on the surface of electrodes. When an antibody-containing solution is loaded, the interaction between antibodies and antigens will cause a change in the impedance of the electrode/electrolyte system, which can be sensed by an external impedance readout system to realize the detection of the antibody–antigen interaction. The impedance between a pair of electrodes immersed in liquid can be approximated as a network of interfacial capacitors and resistive components including charge transfer resistance and electrolyte resistance, as conceptually shown in Fig. 1c. Impedance immunosensors usually exploit the change in thickness or dielectric properties of the dielectric layer at the electrolyte–dielectric interface, which will lead to a change in the (interfacial) capacitance of the device during antibody– antigen interaction. The interfacial capacitor consists of a series connection of equivalent capacitors caused by electrical double layer (EDL) and macromolecule deposition. When a solid material is immersed into an electrolytic solution, the solid surface will acquire surface charges. To maintain charge neutrality, a thin layer of counter ions is formed at the solid/liquid interface to neutralize the surface charges at the solid surface, which is commonly known as the EDL. Electrically, EDL can be modeled as a capacitor. The layers of counter ions and surface charges are equivalent to the two plates in a capacitor, and the plate separation distance is the EDL thickness. When macromolecules are adsorbed onto the electrode surface, the interfacial capacitance Cint will change due to the change in the thickness and surface area of Cint, which can then be used to indicate the deposition of macromolecules or particles on the electrode as well as to correlate with the macromolecule or particle concentration in the uid. As shown in Fig. 2, prior to the affinity assay, the electrode surface is functionalized with a layer of probe molecules (antigens in this work) to achieve specicity for the targeted antibody. The interfacial capacitance can be approximated as 3s Cint ¼ 3s Aint dag þ dedl ; (1) 3p where 3s and 3p are the permittivities of the sample solution and the probe protein (antigen). The relative permittivity of protein
Paper is around 2–3,27 while that of water is 80. Aint is the electrode area, dag is the antigen thickness, and dedl is the EDL thickness, approximated by the Debye length of the buffer solution according to the Debye–Huckel theory.28 During the assay, the sample solution containing the target antibody will be introduced. When the binding of antigen–antibody takes place, the interfacial capacitance Cint is expected to change to 3s 3s dab þ dag þ dedl ; (2) Cint;binding ¼ 3s Aint;binding 3t 3p where 3t is the permittivity of the target protein, and Aint,binding is the effective area of the interfacial capacitor. The change in Cint could be either an increase or a decrease, as conceptually shown in Fig. 2a and b. As a result of the binding, antibodies are deposited onto the surface. The thickness of the dielectric layer could increase, which could cause a decrease in the interfacial capacitance. On the other hand, randomly deposited antibodies could cause an increase in the capacitor's surface area due to extra topology introduced by the antibody, especially when the probe molecules (antigens) are spaced apart, leading to a higher interfacial capacitance, as shown in Fig. 2b. Both changes could occur during the binding. Oen, one type of change dominates over the other, and then the detection of binding is possible. The situation in Fig. 2a, i.e. a decrease in Cint due to antigen– antibody binding, was commonly observed.26 In a diluted buffer solution, the EDL is relatively thick. As EDL envelops the antibodies on the electrodes, ne features on the scale of EDL thickness (dedl) will be lost, and the Cint change will be dominated by an increase in its thickness, i.e. Cint reduces. When EDL thickness is comparable to that of antibody topology, a positive change of Cint is possible, especially in a buffer solution of high ionic strength.29 Our work found that both an increase and a decrease in Cint could possibly arise from antigen–antibody binding, since an increase of Cint was consistently observed in human TB detection and a decrease in bovine TB detection. Preliminary experiments were conducted to support the above hypothesis about the interfacial capacitance change. Buffer solutions of two different ionic strengths, 1 mM and 2 mM phosphate buffer solutions (PBS), were used. The results corroborate that the EDL thickness (nm) relative to the characteristic length of the deposited macromolecules will determine whether a decrease or an increase in interfacial capacitance would result from binding reactions.
Fig. 1 (a) Commercially available electrode chips, i.e. surface acoustic wave (SAW) resonator chips (PARS 433.92, AVX Corp.). The left one was modified for immunosensing. (b) A scanning electron micrograph of the microelectrode array in the SAW chip. (c) Equivalent circuit for a pair of microelectrodes immersed in an electrolyte. Cint: interfacial capacitance, Rct: charge transfer resistance, Rs: electrolyte resistance, Cs: electrolyte capacitance.
7190 | Analyst, 2013, 138, 7188–7196
This journal is ª The Royal Society of Chemistry 2013
View Article Online
Published on 07 October 2013. Downloaded by University of California - Santa Cruz on 22/10/2014 10:32:43.
Paper
Analyst
Fig. 2 Two possible topology changes at the solid/fluid interface due to the protein binding reaction. (a) The thickness of the interfacial layer increases while its surface area decreases, Cint reduces as a result; and (b) when the increase in the surface area of Cint dominates over the changes in its thickness, Cint increases (Ab: antibody; Ag: antigen).
2.2
ACEK acceleration of the binding reaction
For affinity based sensing, the detection of binding event usually relies on the diffusion of target macromolecules to the binding sites. It could take hours or even days for the amount of bound molecules to reach a detectable level.30 Therefore, great effort has been devoted to develop a bioparticle concentration mechanism to shorten the detection time and increase the sensitivity. ACEK, as a particle and uid manipulation mechanism, has minimal requirements on the device fabrication and operation to be incorporated into a detection system – only microelectrodes and their AC signal source need to be added. ACEK effects use an AC electric eld to induce particle and uid movement, so that macromolecules can be in situ concentrated onto microsensors.31 When an inhomogeneous AC electric eld is applied to an aqueous solution, both particle movement and microows can be induced to transport particles. Direct particle movement can be caused by dielectrophoresis (DEP), and particle can also be carried by microows such as AC electroosmosis or AC electrothermal ows to reach the microsensor. The manipulation of particles by DEP is based on the difference between the particle polarizability and that of the medium solution17 at a certain frequency. DEP force on a spherical particle can be expressed as follows: # 3*p 3*m 2 V |E| ; ¼ p3m a Re * 3p þ 23*m "
FDEP
3
(3)
where a is the radius of the particle, and 3*p and 3*m are particle and medium complex permittivities, respectively, given as 3* ¼ 3 + is/u (s: conductivity; u: angular frequency). The term in the bracket, known as the Clausius–Mossotti factor, strongly depends on AC frequency and buffer conditions, and determines the polarity of DEP force. When the Clausius–Mossotti factor is positive, the particle will experience a positive DEP force, moving towards high electric eld regions, such as electrode edges. When the Clausius–Mossotti factor is negative, the particles will experience a negative DEP force, being repelled from electrodes. Positive DEP is employed for particle concentration in this work.
This journal is ª The Royal Society of Chemistry 2013
As it can be seen from eqn (3), the magnitude of DEP force depends on the particle volume. In the case of nanoscale macromolecules, DEP may not be effective unless the molecules are located within a very short distance to the electrodes (
