Biosensors for hepatitis B virus detection

Abstract

A biosensor is an analytical device used for the detection of analytes, which combines a biological component with a physicochemical detector. Recently, an increasing number of biosensors have been used in clinical research, for example, the blood glucose biosensor. This review focuses on the current state of biosensor research with respect to efficient, specific and rapid detection of hepatitis B virus (HBV). The biosensors developed based on different techniques, including optical methods (e.g., surface plasmon resonance), acoustic wave technologies (e.g., quartz crystal microbalance), electrochemistry (amperometry, voltammetry and impedance) and novel nanotechnology, are also discussed.

Key Words: Biosensor, Hepatitis B virus, Diagnosis, Detection, Quartz crystal microbalance

Core tip: This paper reviews the current state of biosensor research with respect to efficient, specific and rapid detection of hepatitis B virus. The biosensors developed based on different techniques, including optical methods (e.g., surface plasmon resonance), acoustic wave technologies (e.g., quartz crystal microbalance), electrochemistry (amperometry, voltammetry and impedance) and novel nanotechnology, are also discussed.

INTRODUCTION

Hepatitis B virus (HBV) infection is one of the major health problems worldwide, and may lead to chronic hepatitis, cirrhosis and primary liver cancer, particularly if the patient’s diet and health conditions are not well controlled[1-3]. It is estimated that there are 2 billion people with a serological profile of past or current HBV infection, and 360 million patients with chronic HBV-associated liver disease[4,5]. Patients are at an especially high risk when their serum viral load is > 10⁵ copies/mL, according to a report by the REVEALHBV (risk evaluation of viral load elevation and associated liver disease/cancer-hepatitis B virus) group[6,7]. Hence, it is important to detect and monitor HBV at an early stage of infection. Hepatitis B is a familiar disease in China, and determination of virus concentration can provide the key foundation for diagnosis and treatment.

Currently, the most commonly used clinical diagnostic methods for HBV detection are immunoassay and polymerase chain reaction (PCR). Immunoassays are based on serological methods that target viral antigens or antibodies, which can present a good selectivity and achieve 100% accuracy. However, the immunoassay method does not provide quantitative results and the detection is limited by serological response[8]. Hepatitis B surface (HBs) antibody is the most commonly used immunoassay target and appears even several months after the acute period of HBV infection[9]. In clinical application, PCR is traditionally the standard technique to identify HBV infection. Quantitative PCR has been used to estimate the initial amount of virus DNA by measuring the amount of amplified product. Real-time PCR is based on the evaluation of the threshold cycle (C_T) when amplification of the PCR product is first detected[10]. PCR consists of 20-40 repeated cycles to achieve a detectable DNA concentration, so it requires efficient control of thermal cycles and thus has higher instrumentation costs. PCR is also known to have errors caused by its hybridization mechanism. Consequently, it is necessary to develop a detection technique that has the characteristics of cost-effectiveness, fast response, portable capability, and high sensitivity compared with traditional clinical diagnostic methods.

A biosensor is an analytical device used for the detection of analytes, which combines a biological component with a physicochemical detector[11]. Recently, an increasing number of biosensors have been used in clinical research, for example, the blood glucose biosensor[12,13]. Most of the clinical research on biosensors was based on immunological reaction or DNA hybridization, and the sensor always gave rapid results with a high sensitivity[14,15]. DNA biosensors based on nucleic acid hybridization are currently under intense investigation owing to their increasing importance in the diagnosis of disease, with low cost and low power requirements[16]. This type of biosensor can be prepared by immobilizing single-stranded DNA probes on different electrodes and physicochemical detectors are used to measure the hybridization events between the DNA probes and their cDNA fragments.

This review looks at the current state of biosensor research with respect to efficient, specific and rapid detection of HBV. The biosensors developed based on different techniques, including optical methods [e.g., surface plasmon resonance (SPR)], acoustic wave technologies [e.g., quartz crystal microbalance (QCM)], electrochemistry (amperometry, voltammetry and impedance) and novel nanotechnology, are also discussed.

BIOSENSORS BASED ON NANOTECHNOLOGY

Recently, with the development of nanotechnology, researchers are constantly expanding the applications of nanotechnology with unique properties to construct novel biosensors. For instance, quantum dots[17], carbon nanotubes[18], nanowires[19] and magnetic nanoparticles[20-22] have attracted much attention as signal transducers. Biosensors based on nanotechnology show high specificity and sensitivity after being labeled with DNA probe or antibody. The nanoparticle-based biosensor has high specificity, ease of operation, low cost, and the sensitivity needed for the rapid and reproducible detection of pathogenic microorganisms in clinical specimens. However, the covalent labeling with nanoparticles is time consuming and involves complicated synthetic procedures.

Gold nanorod material has high light absorption and a large scattering cross-section in the SPR wavelength regions. Therefore, Wang et al[23] designed a novel gold nanorod biosensor based on localized SPR (LSPR) to detect hepatitis B surface antigen (HBsAg). Investigation of the HBs-antibody-modified gold nanorod indicates that physical adsorption and successive blocking lead to a flexible shell enveloping the nanorod, which minimizes the nonspecific adsorption and allows the assays to work in buffer, serum and plasma. The experimental results indicate that a gold-nanorod-based LSPR biosensor is capable of measuring HBsAg concentration even at 0.01 IU/mL, which is about 40 times lower than the limit of detection of the ELISA method. The gold-nanorod-based biosensor can be utilized in quantitative analysis with a dose-dependent response ranging from 0.01 to 1 IU/mL.

ELECTROCHEMICAL DNA BIOSENSORS

Electrochemistry possesses attractive features over other existing measurement systems, therefore, electrochemical biosensors are expected to provide a simple, accurate and inexpensive platform for DNA analysis[24]. Electrochemical biosensors have attracted considerable attention in recent years, and have provided a sensitive, accurate, rapid and cost-effective platform for a variety of practical applications in the fields of medical diagnostics, clinical genetic analysis, forensic identification and environmental monitoring[25,26]. In clinical diagnosis, electrochemical transducers offer attractive advantages, for example, good sensitivity, high specificity, simplicity and low cost, for converting nucleic acid hybridization events into analytical signals[27,28]. The complex formed from the bioreceptor and biomolecular binding at the sensor surface results in a detectable change, converted into a quantitative amperometric, potentiometric or impedimetric signal. Electrochemical transducers are often used for detecting DNA hybridization events, due to their high sensitivity, small dimensions, low cost, and compatibility with microfabrication technology[29].

Voltammetric and amperometric biosensors

Zhang et al[30] have developed an electrochemical DNA biosensor for the detection of HBV DNA fragments. The biosensor relies on the covalent immobilization of the 21-mer single-stranded DNA (ssDNA) related to HBV genes on the modified glassy carbon electrode (GCE). Using (CdL₂)²⁺ as a novel electroactive indicator, the hybridization between the probe and its complementary ssDNA was investigated by differential pulse voltammetry. Experiments with non-complementary oligonucleotides were carried out to assess the selectivity of the developed electrochemical DNA biosensor. The target HBV DNA could be quantified in the range from 1.01 × 10^-8 to 1.62 × 10^-6 mol/L with good linearity (γ = 0.9962). The detection limit was 7.19 × 10^-9 mol/L.

In a study by Ding et al[31] a label-free electrochemical biosensor for the detection of oligonucleotides related to HBV sequences via the interactions of DNA with the redox-active complex 2,9-dimethyl-1,10-phenantroline cobalt [Co(dmp)(H₂O)(NO₃)₂] was described. The study was carried out by the hybridization of a 21-mer probe DNA modified on a GCE with target DNA, and [Co(dmp)(H₂O)(NO₃)₂], which was comparable in size to the small groove of native double-helix DNA, was used as an electrochemical indicator. Under the optimal conditions, the electrical signal had a linear relationship with the concentration of target DNA, ranging from 3.96 × 10^-7 to 1.32 × 10^-6 mol/L, and the detection limit was 1.94 × 10^-8 mol/L (S/N = 3). The biosensor had good selectivity by detecting the three-base mismatch sequence.

Hashimoto et al[32] reported a microfabricated disposable-type electrochemical DNA biosensor, based on photolithography technique, for the detection of HBV genomic DNA. HBV DNA extracted from patients’ sera was quantified in the range of 10⁴-10⁶ copies/mL, and the biosensor showed a good correlation with competitive PCR. The results suggested that the microfabricated disposable DNA biosensor can provide a specific and quantitative detection of HBV DNA in serum.

In a study by Meric et al[33], an electrochemical biosensor for voltammetric detection of DNA sequences related to HBV and TT virus (TTV) from PCR-amplified samples was described. The electrochemical DNA biosensor relied on the immobilization of the 21- or 24-mer single-stranded oligonucleotides (probe), which were related to the HBV and TTV sequences, and hybridization of these oligonucleotides with their complementary sequences (target) at a carbon paste electrode. The extent of hybridization between the probe and target sequences was determined using square wave voltammetry and methylene blue (MB) as a hybridization indicator. With its specific interaction with the guanine bases, the MB signals from the hybrid and the probe can easily be distinguished from each other. The point mutations, which include guanine bases in the DNA sequences, can rapidly be detected using this specific interaction of MB with unbound guanine bases. However, they did not give the detection range and limit for HBV detection.

Impedance biosensors

Electrochemical impedance spectroscopy is a sensitive technique, which monitors the electrical response of the studied system after application of a periodic small amplitude AC signal. System response provides information concerning the interface state (presence of adsorbed species) and can detect the occurrence of interfacial reactions[34,35].

Hassen et al[36] reported a new approach based on DNA hybridization for detecting HBV using an electrochemical impedance biosensor. DNA probes modified with biotin at the 5’ position were immobilized on streptavidin-modified magnetic nanoparticles by biotin-streptavidin interaction. The hybridization reactions with specific cDNA target and non-complementary sequence were investigated by impedance spectroscopy. Results showed that the impedance biosensor allows detection of 50 pmol/L HBV DNA in a sample of 20 μL.

PIEZOELECTRIC BIOSENSORS

Piezoelectric biosensors are mass-sensitive and their resonant frequencies are directly dependent on the mass of the sensing layer, as well as the captured external biomolecules. Therefore, they have been developed for label-free monitoring of affinity interactions between molecules with real-time output, high sensitivity and good specificity[3,37,38]. Most of the reported piezoelectric biosensors utilize quartz crystal disks as transducers[39,40], and target DNA is normally the target analyte. However, these devices are subject to time-consuming procedures and inability for multi detection due to the physical nature of the quartz crystal.

QCM biosensors

QCM is based on the property of an AT cut quartz crystal that oscillates when an alternating voltage is applied. The quartz surface always has a thin gold (or other) metal layer deposited onto it, which doubles as a conductor for the AC current flowing and also provides an attachment surface for a bioreceptor to be immobilized (Figure 1). This deposition can be monitored by the frequency decrease caused by the additional mass on the metal layer that is related to the mass change via the Sauerbrey equation[41]. Recently, increasing attention has been paid to the development of QCM biosensors because of their many merits such as compact size, high sensitivity and good specificity, low cost, label-free detection, and rapid response[3,42]. Moreover, advances in biosensors make multiple-analyte diagnosis possible, which may eliminate expensive and time-consuming detection procedures in clinical diagnosis.

Figure 1 Schematic representation of constructed crystal for quartz crystal microbalance biosensor.

Xu et al[43] have developed a diaphragm-based QCM immunoassay chip to detect simultaneously HBV and α-fetoprotein (AFP) antibodies. The total assay time was < 2 h. The frequency shift-based calibration curves showed a detection limit of 0.1 ng/mL and a dynamic detection range of 0.1-10 000 ng/mL for both anti-HBsAg and anti-AFP. The small frequency variations of the reference sensors during the whole detection processes show that the washing processes and nonspecific biomaterials absorption have negligible effects on the sensor resonant frequency. The results demonstrate that the piezoelectric immunoassay chip has potential applications for rapid, specific, sensitive, and multiple detection of HBV.

Yao et al[39] have used peptide nucleic acid (PNA) probes instead of DNA probes to construct PNA-QCM biosensors for real-time monitoring of the hybridization assay of HBV. The PNA probe can combine target sequences more effectively and specifically than DNA probes. The PNA probe is designed and immobilized on the surface of the biosensor to substitute the conventional DNA probe for direct detection of HBV genomic DNA without previous amplification by PCR. The hybridization assay is completed within 50 min. The detection limit is 8.6 pg/L and the clinical specificity is 94.44% compared with real time-PCR. The PNA probe can distinguish sequences that differ in only one base. Detection sensitivity can be improved and detection time can be reduced by adding RecA-protein-coated complementary ssDNA, which complements HBV gene regions.

Rolling circle amplification (RCA) is an isothermal amplification technique for small circular DNA templates, with the unique property of product localization[44]. Yao et al[45] have described the application of RCA-based QCM biosensors for direct detection of HBV genomic DNA from clinical samples. The covalent bonding between the capture probes and the gold electrode surface guarantees that the linkage between the capture probes and the amplified RCA products is maintained during the assay. Making use of the high amplification efficiency of Phi29 DNA polymerase and the remarkable precision of Escherichia coli DNA ligase in differentiating mismatched bases at the ligation site, we can detect as few as 10⁴ copies/mL HBV DNA. The experimental results show that RCA has significantly enhanced sensitivity for the target strand compared to the single-base mismatch strand. With 60 min RCA duration, the proposed detection shows over 10 times amplification of the original signals.

In a study by Zhou et al[40] a highly sensitive piezoelectric DNA biosensor has been developed based on the sensitive mass-transducing function of the QCM and the specificity of the nucleic acid hybridization reaction. An HBV nucleic acid probe was immobilized onto the gold electrodes of a 9-MHz AT-cut piezoelectric quartz crystal by the polyethyleneimine adhesion, glutaraldehyde cross-linking or physical adsorption method. The frequency shifts of hybridization have a better linear relationship with the amount of HBV DNA, when the amount is 0.02-0.14 μg/mL.

Microcantilever biosensors

Microcantilevers have emerged as viable biosensors, demonstrating prominent performance[46,47]. Recently, the structures and materials of microcantilevers have been improved due to advances in the micro-/nano-electromechanical system, and the applications of microcantilevers have been significantly expanded due to their utilization in the nano-/biotechnology field. Microcantilever biosensors have several advantages over the aforementioned methods, such as rapid detection, simple equipment operation, low cost, and label-free detection within complex biological serum. Thus, microcantilever biosensors have been used successfully for DNA hybridization, immunoassays and particle detection including viruses, bacteria and cells[48,49].

Huang et al[50] have developed a DNA-sensing device based on microcantilevers. The experimental results revealed that the DNA detection system-on-a-chip (SoC) has good selectivity between 1-bp-mismatched DNA and all-matched DNA. Utilizing the N⁺ polysilicon 2 embedded layer as a piezoresistor, the stress induced by the microcantilever-beam deflection can be measured. The developed SoC was the first fully integrated monolithic microcantilever biosensor system, which includes sensors, interface circuits, digital processor, and wireless communication on a single chip for HBV DNA detection. The experimental results showed that the HBV DNA SoC can successfully distinguish the signal of all-mismatched, 1-bp-mismatched, and all-matched sequences of 19-mer HBV-conserved DNA sequences. At the same time, the concentration detection range has been identified from 1 pmol to 10 nmol. That was the first time that the HBV DNA detection capability has been demonstrated by a fully integrated wireless SoC. The microcantilever SoC offers the characteristics of real-time and label-free detection with high sensitivity and selectivity.

Cha et al[51] have reported HBV DNA detection using a silica nanoparticle-enhanced dynamic microcantilever biosensor. A 243-mer nucleotide of HBV DNA precore/core region was used as the target DNA. For detection purposes, the capture probe on the microcantilever surface and the detection probe conjugated with silica nanoparticles were designed specifically for the target DNA. HBV target DNAs of 23.1 fmol/L to 2.31 nmol/L, which are obtained from PCR products, are detected using a silica nanoparticle-enhanced microcantilever. The HBV target DNA of 243-mer is detected down to the picomolar level without nanoparticle enhancement and femtomolar level using a nanoparticle-based signal amplification process. There is an almost 2-3 orders of magnitude increase in sensitivity, and the microcantilever is a highly sensitive and reliable diagnostic tool for DNA detection.

Surface acoustic wave biosensors

Surface acoustic wave (SAW) sensors, which are a type of piezoelectric sensor, were first introduced by White and Voltmer, and are potential biochemical sensing platforms with excellent sensitivity, speed and reliability. Since Wohltjen and Dessy’s application, SAW sensors based on Rayleigh surface waves have been used as highly sensitive sensors[52].

Lee et al[53] have demonstrated an application of low-wave mode SAW immunosensors to detect HBs antibody in aqueous conditions. The resonance frequency shift has been monitored to detect specific binding of HBs antibody to the immobilized HBsAg. The sensor shows binding specificity to HBs antibody and a linear relationship between the frequency shift and antibody concentration with a sensitivity of 0.74 Hz/(pg/μL) and a detection limit < 10 pg/μL. The SAW immunosensor can successfully detect HBs antibody in whole blood samples without any pretreatment.

SPR BIOSENSORS

The SPR method is based on total internal reflection in a glass prism onto which a thin metal film (usually gold) is deposited. When total internal reflection of the incident light occurs, an evanescent wave is set up that exerts a field outside the prism. This can only occur when the film is thin enough, about 50 nm, as the delocalized electrons within the metal cause a surface plasmon to be established at the metal-ambient interface. This causes a wave to propagate along the surface and therefore reduces the intensity of light reflected[41]. The advantages of SPR biosensors are the direct detection, without any additional labeling, and the possibility of online monitoring of affinity interactions[54]. The SPR method is popular because it allows label-free, high-sensitivity, real-time analysis and flexible system design. For example, the design of various orderly nanostructures has been shown to provide strong local electrical field enhancement, allowing a higher sensitivity and a wider dynamic range[37,55]. SPR sensor device design is trending toward miniaturization, low cost and user friendliness[56,57] (Figure 2).

Figure 2 Schematic representation of surface plasmon resonance biosensor. Incidence light illuminates thin gold film, causing electrons to be excited and form a surface plasmon wave.

To develop a high-sensitivity, low-cost screening system for HBV detection, Chuang et al[58] demonstrated the detection of the LAMP reaction by the SPR analytical method without immobilizing probes. The HBV template mixed in 10 μL LAMP solution could be detected using the SPR-LAMP system within 17 min, even at the detection-limited concentration of 2 fg/mL. They also analyzed the correlation coefficients between the initial concentrations of HBV DNA templates and the system response (ΔRU) at different amplification times to establish an optimal endpoint of 25 min (R² = 0.98).

Chung et al[9] have described the use of SPR to detect captured human HBV antibodies using an HBsAg in conjunction with a secondary antibody. The detection limit was found to be 10 nmol HBV antibody. The detection limit of the SPR biosensor for the medical diagnosis was similar to the commercial ELISA kit.

TRANSLATIONAL PERSPECTIVES

In recent years, biosensors have been shown to provide complementary and additional information contributed by the well-established bioanalytical techniques. Particular advantages of biosensors are as follows: the possibility of minimalizing the setup, in principle down to the molecular scale; the use of well-established microsystem technologies during manufacture (of at least certain sensor components); integration of signal preprocessing steps on a chip; and building arrays for more complex analysis. Detecting viruses and viral components using biosensors is a relatively new concept and still in its infancy. Traditional viral detection methods are time consuming and expensive, and consequently, demand for accurate viral biosensors by rapid detection systems is increasing. In most cases, only limited quantities of HBV are available from clinical samples. In most biosensor studies, both the sensitivity and specificity of the HBV detection were unaffected by serum samples (Table 1). However, it is necessary to point out that more detailed work is required before the clinical application of biosensors.

Table 1 Biosensor techniques discussed in this review.

Detection target	Characteristics of biosensor	Detection limit	Ref.
HBsAg	Nanorods biosensor	0.01 IU/mL	[23]
HBsAb	QCM	0.1 pg/μL	[43]
	SAW	10 pg/μL	[53]
	SPR	10 nmol/L	[9]
HBV DNA	Electrochemical biosensor	7.19 × 10-9 mol /L	[30]
		1.94 × 10-8 mol /L	[31]
		1.77 × 10-14 mol /L	[32]
		2.5 × 10-6 mol /L	[36]
	QCM	8.6 × 10-12 g/L	[39]
		1.77 × 10-14 mol/L	[45]
		2 × 10-8 g/L	[40]
	Microcantilever biosensor	10-12 mol/L	[50]
		2.31 × 10-14 mol/L	[51]
	SPR	2 × 10-12 g/L	[58]

HBsAg: Hepatitis B surface antigen; HBsAb: Hepatitis B surface antibody; HBV: Hepatitis B virus; QCM: Quartz crystal microbalance; SAW: Surface acoustic wave; SPR: Surface plasmon resonance.

The development of isothermal amplification technology, such as strand displacement amplification and RCA, makes the amplification of nucleic acid sequences without PCR possible, which provides an opportunity for biosensor miniaturization. RCA is an isothermal amplification technique that is adaptable to the on-chip signal amplification format. Unlike other amplification procedures, RCA produces a single amplified product that remains linked to the DNA primer. Circularization of the padlock probes in the RCA reaction is specific in that 1-bp mismatch can be specifically discriminated[59-62]. As a result of its simplicity, specificity and high sensitivity, RCA has attracted considerable attention in biosensor detection. Aptamers are a new class of molecules with great potential to rival monoclonal antibodies in therapeutic, diagnostic and analytical, as well as basic research applications. In comparison to antibodies as capture molecules in biosensors, aptamers have several advantages. Aptamers are produced in vitro, and their binding affinity, specificity, and stability can easily be manipulated and improved by rational design. At the same time, aptamers can be modified with functional groups to allow covalent or direct immobilization on biochips, resulting in highly ordered receptor layers that can improve detection sensitivity significantly[63,64]. As stable RNA or DNA oligonucleotides, aptamers can be denatured and refolded many times without loss of activity[65]. The characteristics and low cost of synthesis make aptamers particularly suitable for biosensor detection.

Since their development in the 1990s, SPR and QCM have been the most researched techniques to date. However, the novel biosensors will be more attractive with the progress of isothermal amplification technology and aptamer techniques. We hope that a portable, label-free biosensor for the sensitive and specific HBV detection will be available in the near future.