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Hepatitis B virus (HBV) still remains a major global public health problem. One-half to one-third of the total HBV infected people died due to late detection of HBV. Serological antigen and viral HBV detections can help in the diagnosis, referral, and treatment of HBV. Available methods for HBV detection mostly used bulky instruments. Miniaturization of devices for HBV detection has been started by narrowing down the size of the devices. Several methods have also been proposed to increase the selectivity and sensitivity of the miniaturized methods, such as sandwich recognition of the biomarkers and the use of nano- to micro-sized materials. This review presents recent HBV detections in the last two decades from laboratory-based instruments towards microfluidic paper-based analytical devices (µPADs) for point-of-care testing (POCT) purposes. Early and routine analysis to detect HBV as early as possible could be achieved by POCT, especially for areas with limited access to a central laboratory and/or medical facilities.

In nucleic acid testing (NAT), gold nanoparticle (AuNP)-based lateral flow assays (LFAs) have received significant attention due to their cost-effectiveness, rapidity, and the ability to produce a simple colorimetric readout. However, the poor sensitivity of AuNP-based LFAs limits its widespread applications. Even though various efforts have been made to improve the assay sensitivity, most methods are inappropriate for integration into LFA for sample-to-answer NAT at the point-of-care (POC), usually due to the complicated fabrication processes or incompatible chemicals used. To address this, we propose a novel strategy of integrating a simple fluidic control strategy into LFA. The strategy involves incorporating a piece of paper-based shunt and a polydimethylsiloxane (PDMS) barrier to the strip to achieve optimum fluidic delays for LFA signal enhancement, resulting in 10-fold signal enhancement over unmodified LFA. The phenomena of fluidic delay were also evaluated by mathematical simulation, through which we found the movement of fluid throughout the shunt and the tortuosity effects in the presence of PDMS barrier, which significantly affect the detection sensitivity. To demonstrate the potential of integrating this strategy into a LFA with sample-in-answer-out capability, we further applied this strategy into our prototype sample-to-answer LFA to sensitively detect the Hepatitis B virus (HBV) in clinical blood samples. The proposed strategy offers great potential for highly sensitive detection of various targets for wide application in the near future.

DNA analysis is essential for diagnosis and monitoring of many diseases. Conventional DNA testing is generally limited to the laboratory. Increasing access to relevant technologies can improve patient care and outcomes in both developed and developing regions. Here, we demonstrate direct DNA analysis in paper-based devices, uniquely enabled by ion concentration polarization at the interface of patterned nanoporous membranes in paper (paper-based ICP). Hepatitis B virus DNA targets in human serum are simultaneously preconcentrated, separated, and detected in a single 10 min operation. A limit of detection of 150 copies/mL is achieved without prior viral load amplification, sufficient for early diagnosis of hepatitis B. We clinically assess the DNA integrity of sperm cells in raw human semen samples. The percent DNA fragmentation results from the paper-based ICP devices strongly correlate (R(2) = 0.98) with the sperm chromatin structure assay. In all cases, agreement was 100% with respect to the clinical decision. Paper-based ICP can provide inexpensive and accessible advanced molecular diagnostics.

A novel three-dimensional DNA-AuNPs network structure amplification strategy was employed to design a lateral flow biosensor by introducing streptavidin coated gold nanoparticles (Au-SA) in this paper. They act as amplification probes which aggregate numerous gold nanoparticles (AuNPs) on test line by forming a three-dimensional DNA-AuNPs network structure in the presence of target. Sensitive detection of nucleic acid with point-of-care analysis is significant for infectious agent, early diagnosis and treatment of genetic diseases. The use of these particles in rapid ultrasensitive point of care (POC) lateral flow assays lead to a linear range from 0.1pM to 250nM with a limit of detection of 0.01 pM without polymerase chain reaction (PCR). The proposed method could increase the sensitivity by 4 orders of magnitudes than traditional sandwich assays labeled with AuNPs. Furthermore, the assay owns good reproducibility and stability, which will prove practical diagnostic applications.

Aims—To compare the efficiency of five nucleic acid extraction kits for processing clinical material for the diagnosis of infection. Methods—Five nucleic acid extraction kits for processing clinical material for the diagnosis of infection were compared for their relative efficiencies in purifying and recovering either viral DNA or RNA from serum samples. Quantitative polymerase chain reaction (PCR) assays for hepatitis B virus (HBV) DNA and hepatitis C virus (HCV) RNA were performed on the LightCyclerTM instrument to determine the relative concentrations of the viral nucleic acids recovered by the various protocols. Results—Large differences between kits in recovery efficiencies were observed for HBV DNA, with those protocols using enzyme digestion in addition to chaotropic solutions performing better than those using chemical disruption alone. Conclusions—There were large differences between the kits and it appeared that those extraction kits containing a proteolytic enzyme and carrier nucleic acid (and that are supplied RNase free) have the widest potential application in the routine microbiology laboratory.

We present a low cost, simple and integrated device for medical diagnostics in low-resource settings called the lab-in-a-pen. Finger pricking, and sample collection and processing, are integrated with commercially available paper-based assays in a pen format. This approach ensures safety (i.e. biological sample and sharps containment) and can be used by untrained end users across multiple settings. The pen format also leverages existing low cost, high volume manufacturing and assembly methods. We characterize sample wicking in the lab-in-a-pen using porcine whole blood. The clinical diagnostic utility and usability of the lab-in-a-pen is established by testing of patients for Hepatitis B surface antigen (HBsAg) and Hepatitis B 'e' antigen (HBeAg) by medical staff at the National Hospital for Tropical Diseases in Hanoi, Vietnam.

In order to determine the relationship between hepatitis B virus (HBV) infection of human white blood cells and different forms of HBV-associated liver diseases, we tested for HBV DNA in the sera and leukocytes of 11 healthy individuals without any serological markers of HBV infection and 91 patients with HBV infection and other gastrointestinal and urinary diseases by dot and Southern blot hybridization. HBV DNA was found in leukocytes of chronic HBV carriers, in acute and chronic hepatitis, and in patients with liver cirrhosis and hepatocellular carcinoma. Between 27 and 50% of individuals in different categories of patients examined were positive for leukocyte HBV DNA. HBV DNA was also detected in the sera of some of these patients but was absent in others. Serum HBV DNA-positive rates seemed to be highest in hepatitis B e antigen-positive asymptomatic carriers (8/10, 80%), and tended to drop to lower levels as the disease progressed to liver cirrhosis (0/8) while leukocyte HBV DNA-positive rates were highest in patients with cirrhosis (4/8, 50%). The results also show that in individuals who were serologically negative for hepatitis B surface antigen (HBsAg) and positive for antibodies to HBsAg and/or HBcAg, HBV DNA was absent in most of the sera (27/28, 96%) but it was present in leukocytes of some of these patients (7/28, 25%). In control experiments with 11 healthy individual, HBV DNA was not detected in either sera or leukocytes. In all the cases with leukocyte HBV DNA, the HBV DNA molecules were present in free forms with discrete sizes. The exceptions were a case of liver cirrhosis and a case of chronic hepatitis with possible HBV sequence integration into high molecular weight cellular DNA. Since HBV does infect human leukocytes, it may perhaps interfere with the immunological functions of the white blood cells, and thus play an important role in the pathogenesis of HBV-induced liver disease.

The hepatitis B surface antigen (HBsAg) is highly immunogenic and induces an antibody response which is protective in vivo against hepatitis B virus (HBV) infection. Human monoclonal antibodies specific for HBsAg were produced, which could have potential therapeutic applications. Lymphocytes obtained from a vaccinated donor were stimulated in vitro and fused with the human myeloma cell line GM 4672, and eight hybridomas were obtained. Three of these clones, which reacted in an ELISA against the HBsAg vaccine, were expanded, subcloned and further analyzed. The subclones E7C2, C4C10, and D5B2 were able to bind to different HBsAg preparations, which express various subtypes, and recognized the major HBsAg peptides in Western blot analysis. Cross-inhibition experiments showed that E7C2, C4C10 and D5B2 are directed against the same epitope and have an affinity constant ranging from 5 X 10(7) to 3.3 X 10(9) M. Furthermore, these antibodies stained the surface and cytoplasm of the HBsAg-secreting cell lines PLC/PRF/5 and 4.10. The production of immunoglobulins varies from 0.3 to 1.3 micrograms/ml/10(6) and has remained stable over a period of 8 months. These human monoclonal antibodies, which appear to be directed against an antigenic determinant common to all HBsAg subtypes, could be useful in the study of HBV-related liver diseases as well as in their diagnosis and experimental therapy.

Nitrocellulose-protein blotting of serum electrophoresed in agarose gels has been adapted for the study of hepatitis B surface antigen (HBsAg). 125I-labeled anti-HBs was used as the antigen probe, and the electrophoretic migration was monitored by autoradiography. The method required 3 microliter or less of serum and could detect as little as 1 pg of purified HBsAg. Typically, we observed two bands of HbsAg; a moving band which migrated about one-third the distance moved by human serum albumin and a non-migratory band which remained at the loading site. Some examples of the use of the method include: (1) empirical methods for correlating HBsAg concentration in serum to film darkness; (2) observations of mobility changes in serial sera from dialysis patients with chronic HBsAg antigenemia; and (3) detection of related antigens such as antigen from the PLC/PRF/5 hepatoma tissue culture line and the cross-reacting woodchuck patients hepatitis virus surface antigen (WHsAg).