Gold nanoparticles and hepatitis B virus

Abstract Hepatitis B virus (HBV) infection is one of the major health issues in the world presently with high tendency of leading to hepatocarcinoma, cirrhosis, and liver cancer, especially if not properly managed. It has been estimated that there are about 2 billion people with a serological profile of HBV infection, and 360 million patients worldwide living with chronic HBV-associated liver disease, hence the need to find an efficient and precise diagnosis technique to drive a robust treatment for Hepatitis B virus cannot be over emphasized. The emergence of analytical device like biosensor which combines biological and physicochemical element to detect HBV in screened samples has been very helpful in providing a timely intervention to tame this virus. This review focuses on the current state of biosensor researches with respect to various in-depth application of gold nanoparticle for the detection of hepatitis B virus (HBV).


Introduction
Hepatitis B is a worldwide acknowledged public health issue with estimated 360 million infections, 600,000 deaths in addition to being a leading cause of high amount of infant and early childhood morbidity and mortality yearly [1]. According to a 2012 survey report, about 800 million people are living with the virus while in China alone, the carrying rate of the surface antigen is 9.7% [2]. This deadly disease is caused by a DNA virus called hepatitis B virus (HBV) which belongs to the hepadnaviridae family. Humans are the only known natural host and primarily resides and replicates in the liver cells thereby causing either acute or chronic damages to the liver cells leading to deadly liver cirrhosis or hepatocellular carcinoma. The virus is also found in various amount in circulating blood, saliva, breast milk, secretions of semen and vagina [3,4]. HBV is transmitted sexually, mother to child at point of birth and via unprotected contacts with contaminated body fluids and objects such as needle stick. Fortunately, the burden of this lethal infection has been significantly reduced with the successful introduction of vaccines whose debut was recorded as far back as 1981 in the United States [5]. This breakthrough was further complemented with the coming of advanced testing, diagnostic and screening tools and devices. As such there is a pertinent need to devise a simple, fast but yet more efficient technique for the diagnosis of HBV [6]. In the past few decades, there has been an increasing number of the application of biosensors in detecting various pathogens including hepatitis virus [7]. According to it working principle, biosensors are highly sensitive analytical devices made up of a biological element for detection or recognition of a specific analyte based on it inherent physicochemical characteristics and an electronic or optical transducer for signal transmission and measurement [8,9]. As reported by Haasnoot et al. majority of the biomedical research on biosensors have been focused on the immunological reaction or DNA hybridization techniques, and the bio-sensor were consistently efficient in giving results with a high degree of sensitivity [10]. In the light of the rapidly accumulating knowledge and applicability of nanotechnology, researchers have been exploring the unique optical and bio-conjugatory features of gold nanoparticles in pathogen diagnosis and biomolecule detection devices [11]. Table 1 summarizes the available application of biosensor in detecting hepatitis sensor.
Gold nanoparticles have been instrumental in the detection of various pathogens apart from hepatitis virus (Table 2), however this review focuses mainly on the use of gold nanoparticle as a biosensor for detecting HBV gene and antigen [12]. It is important to state here that there have been some reports on other methods developed for the detection of HBV such as single strand conformation [13] and high performance liquid chromatography [14].

Gold nanoparticles as a biosensor and detector
In the past few years, gold nanoparticles have been influential in the detection of DNA because of its unique optical and bio-conjugation features which early works were pioneered by Mirkin et al. paving way for several other studies such as use of spherical gold nanoparticles targeted at detecting DNA [15][16][17]. Lately research attention has shifted to the elongated rod-like gold nanoparticules called gold nanorods (AuNRs). AuNRs unlike other forms, gold particles possesses superior absorption and stronger light scattering properties thereby producing dual absorption peaks in the visible region and near the infrared regions which have been designated as the transverse and longitudinal surface plasmon bands, respectively [18]. AuNRs have been reported to be applicable in biosensor [19] gene delivery [20] and photothermal therapy [21,22]. Specifically Darbha et al. attempted exploring the nonlinear optical features of AuNRs to screen HIV-1 viral DNA sequence [23,24] while Parab et al. worked on pathogen detection with the aid of AuNRs-based optical DNA biosensor [25]. Table 3 presents a brief on some applications of gold nanorods (AuNRs) biosensors in target DNA detection on the basis of its unique optical features.

Application of gold nanoparticle for hepatitis C virus (HCV rna) sensing
A number of research works have established that gold nano-particles based devises can be used to detect hepatitis C virus. For instance, Griffin et al. conceived and developed a procedure known as size and distance dependent nanoparticle surface energy transfer technique for the purpose of HCV RNA sensing and detection [26]. In brief, the underlining principle of the technique shows that RNA probe is branded with fluorophores and absorbed onto the surface of the gold nano-particles such that once the probe binds to the targeted RNA, the dsRNA complex is released into the solution and a fluorescence emission is reestablished. It is important to note that the resultant solution undergo a color change from red to blue due to aggregation of gold nano-particles ( Figure 1). The fluorescence intensity is directly proportional to the concentration of the target RNA in the solution hence could be utilized in determining the quantity of HCV RNA [26]. In a related development, Glynou et al. fabricated a dryreagent strip biosensor for target DNA detection using AuNPs as visual probe. The results obtained compares favorably with no marked differences between it and COBAS AMPLICORTM, a proven and an efficient kit. Further to it merit, the test is guileless with a reported turnaround time of approximately 10 min [27].

Application of gold nanoparticle in detecting HB surface antigen
There have been quite few research studies that demonstrated the application of gold nano-particles in detecting hepatitis B virus [28]. Gold nano-rod (GNR) have been used to develop biosensor for detecting hepatitis B surface antigen (HBsAg) in biological sample or specimen such as buffer, blood serum and plasma [29]. HBsAg is considered the most crucial biomarker for the lab-based diagnosis of hepatitis B virus. The presence of HBsAg in the blood or serum sample signifies a   [50] Mutations in EGFR gene in genomic DNA [51] SNPs in b-thalassemia gene in genomic DNA-mediated by PCR [52] Hepatitis C virus RNA [53] BCR-ABL fusion transcript in clinical samples [54] Fluorescence-based Gold Plasmodium falciparum heat shock protein in infected blood cultures [55] Electric/Electrochemical Gold Human and mouse IgG antibody in human and mouse serum [56] Interleukin-8 (IL-8) cancer biomarker in human serum [57] Interleukin-6 (IL-6) cancer biomarker in calf serum [58] Tumor necrosis factor a (TNF-a) in human serum [59] Anti-hepatitis B virus antibodies and human IgG in human serum[60] SERS (surface-enhanced Raman scattering) Gold Nicotinic acid adenine dinucleotide phosphate (NAADP) [61] in cell extracts Multiple pathogen DNA in clinical specimens [62] NIR (near infrared region) Gold Lymph nodes in mouse [63] Brain vessels in mouse [64] HER2 cancer biomarker in breast adenocarcinoma cells [65] This chronic and acute level of hepatitis B virus infection [30,31]. The operating principle involves labeling the GNR surface with a monoclonal HBsAb to detect HBsAg and the technique was validated via ELISA [32]. Integrating the antibody with concentrated GNRs through nonspecific physisorption lead to the synthesis of the antibody-GNR complex (Figure 2) called the biosensor, which can characterize target proteins. Increase in the concentration of DNA probe in the AuNRs suspension is proportional to a decrease in the fluorescence intensity of FAM as a result of the Fluorescence resonance energy transfer (FRET) which spans from FAM to AuNRs. Also when the complementary DNA (cDNA) is introduced into the system, a further decrease in the fluorescence intensity of FAM is observed due to increase in both electrostatic interaction and FRET effectiveness. Ideally, the regression of the fluorescence intensity of FAM correlated linearly to the concentration of the cDNA with values ranging from 0.045 to 6.0 nmolÁL À1 , and the expression of HBV gene via polymerase chain reaction (PCR) was successfully carried out [11,33].

Application of gold nanorod and polymerase chain reaction in detecting hbv gene
According to a study conducted by Xiaocui et al. which successfully synthesized gold nano-rods (AuNRs) in aqueous solution in accordance to the seed-mediated method as reported by Murphy et al. [34,35]. The procedure begins with preparation of the seed solution done by mixing Cetyltrimethylammonium bromide (CTAB) and HAuCl4 with ice-cold NaBH4 [36] rat. After 2 h, the obtained seed solution is then used for the synthesis of AuNRs. CTAB was carefully mixed with AgNO3 and HAuCl4, while 140 lL of ascorbic acid  [66] with copyright permission. Figure 1. Gold nanoparticle-based assay to detect hepatitis C virus RNA. In hepatitis C virus (HCV)-positive specimens, the fluorophore-labeled probe hybridizes to the target HCV RNA and fluorescence is be detected. In addition, the color of the solution will change from red to blue, owing to the aggregation of AuNPs (a qualitative colorimetric signal indicating the presence of HCV RNA). AuNP: Gold nanoparticle; NSET: Nanoparticle surface-energy transfer. This image adapted from Griffin et al. [26] with copyright permission.
was added to the resultant solution. AuNRs growth is initiated with the addition of 24 lL of the seed solution to the growth solution and kept at 30 C for a day. Finally, the excess CTAB is removed from the solution via centrifugation and AuNRs produced are re-dispersed in distilled water and characterization and morphology of the synthesized gold nano-rods were carried out using TEM (Figure 3).  Figure 4 below in a step-by-step preparation.
In an attempt to further elucidate the immunoreaction between the immobilized HBsAb and HBsAg in the blood sample using the fabricated immune-sensor, Jian-Ding et al. used the differential pulse voltammetry (DPV) approach. The recorded quantitative measurement was determined from 0.2 to 0.68 Volts with pulse amplitude of 50 mV. DPV peak currents were found to decrease with an increase in the value of HBsAg concentration ( Figure 5) thus were interpreted as the amperometric response which decreases with increase in HBsAg concentration [38].
The fabricated electro-chemical immunosensor was shown as been capable of determining HBsAg in human serum (6 samples) and the results obtained were compared to those gotten via ELISA technique. The relative deviations between the two techniques were in the range of À5.76 to þ6.12%, Figure 2. Showing pictorial representation of the synthesis of antibody-functionalized GNRs and the detection mechanism for the biosensor immunoassay in capturing targets in different matrixes. This image adapted from Wang et al. [29] with copyright permission. which suggested that the results of two techniques were almost the same. Conclusively, the new invented AuNP based immunosensor might be a promising tool for determining the HBsAg in human serum clinically.

Conclusions
AuNRs-based fluorescent biosensor has been demonstrated to detect hepatitis B virus DNA. The fabricated biosensor was efficient in detecting complementary DNA sequences and most importantly it successfully detected PCR product of the HBV gene.
This newly developed bio-sensing strategy based on combination of PCR (for specific target gene amplification before detection) with FRET technique could successfully detect HBV or HCV gene from human sample, plasma sample in a concentration range of 0.045 to 6.0 nmolÁL À1 . In addition electrochemical DNA biosensor based on GNRs on a gold-electrode surface has also been demonstrated for detecting HBV, furthermore AuNP based immunosensor was used successfully to detect HBsAg in human serum. However more clinical research is still needed to increase the concentration range and detective and sensitivity of all these AuNP based sensors.

Disclosure statement
The authors deny any conflict of interest in any terms or by any means during the study.