Recent Advances in Surface Plasmon Resonance Imaging Sensors.

The surface plasmon resonance (SPR) sensor is an important tool widely used for studying binding kinetics between biomolecular species. The SPR approach offers unique advantages in light of its real-time and label-free sensing capabilities. Until now, nearly all established SPR instrumentation schemes are based on single- or several-channel configurations. With the emergence of drug screening and investigation of biomolecular interactions on a massive scale these days for finding more effective treatments of diseases, there is a growing demand for the development of high-throughput 2-D SPR sensor arrays based on imaging. The so-called SPR imaging (SPRi) approach has been explored intensively in recent years. This review aims to provide an up-to-date and concise summary of recent advances in SPRi. The specific focuses are on practical instrumentation designs and their respective biosensing applications in relation to molecular sensing, healthcare testing, and environmental screening.

Prevalence and genetic diversity analysis of human coronaviruses among cross-border children.

BACKGROUND: More than a decade after the outbreak of human coronaviruses (HCoVs) SARS in Guangdong province and Hong Kong SAR of China in 2002, there is still no reoccurrence, but the evolution and recombination of the coronaviruses in this region are still unknown. Therefore, surveillance on the prevalence and the virus variation of HCoVs circulation in this region is conducted. METHODS: A total of 3298 nasopharyngeal swabs samples were collected from cross-border children (<6 years, crossing border between Southern China and Hong Kong SAR) showing symptoms of respiratory tract infection, such as fever (body temperature > 37.5 °C), from 2014 May to 2015 Dec. Viral nucleic acids were analyzed and sequenced to study the prevalence and genetic diversity of the four human coronaviruses. The statistical significance of the data was evaluated with Fisher chi-square test. RESULTS: 78 (2.37%; 95%CI 1.8-2.8%) out of 3298 nasopharyngeal swabs specimens were found to be positive for OC43 (36;1.09%), HKU1 (34; 1.03%), NL63 (6; 0.18%) and 229E (2;0.01%). None of SARS or MERS was detected. The HCoVs predominant circulating season was in transition of winter to spring, especially January and February and NL63 detected only in summer and fall. Complex population with an abundant genetic diversity of coronaviruses was circulating and they shared homology with the published strains (99-100%). Besides, phylogenetic evolutionary analysis indicated that OC43 coronaviruses were clustered into three clades (B,D,E), HKU1 clustered into two clades(A,B) and NL63 clustered into two clades(A,B). Moreover, several novel mutations including nucleotides substitution and the insertion of spike of the glycoprotein on the viral surface were discovered. CONCLUSIONS: The detection rate and epidemic trend of coronaviruses were stable and no obvious fluctuations were found. The detected coronaviruses shared a conserved gene sequences in S and RdRp. However, mutants of the epidemic strains were detected, suggesting continuous monitoring of the human coronaviruses is in need among cross-border children, who are more likely to get infected and transmit the viruses across the border easily, in addition to the general public.

Identification of microRNAs in Throat Swab as the Biomarkers for Diagnosis of Influenza.

BACKGROUND: Influenza is a serious worldwide disease that captures global attention in the past few years after outbreaks. The recent discoveries of microRNA (miRNA) and its unique expression profile in influenza patients have offered a new method for early influenza diagnosis. The aim of this study was to examine the utility of miRNAs for the diagnosis of influenza. METHODS: Thirteen selected miRNAs were investigated with the hosts' throat swabs (25 H1N1, 20 H3N2, 20 influenza B and 21 healthy controls) by real-time quantitative polymerase chain reaction (RT-qPCR) using U6 snRNA as endogenous control for normalization, and receiver operating characteristic (ROC) curve/Area under curve (AUC) for analysis. RESULTS: miR-29a-3p, miR-30c-5p, miR-34c-3p and miR-181a-5p are useful biomarkers for influenza A detection; and miR-30c-5p, miR-34b-5p, miR-205-5p and miR-449b-5p for influenza B detection. Also, use of both miR-30c-5p and miR-34c-3p (AUC=0.879); and miR-30c-5p and miR-449b-5p (AUC=0.901) are better than using one miRNA to confirm influenza A and influenza B infection, respectively. CONCLUSIONS: Given its simplicity, non-invasiveness and specificity, we found that the throat swab-derived miRNAs miR-29a-3p, miR-30c-5p, miR-34b-5p, miR-34c-3p, miR-181a-5p, miR-205-5p and miR-449b-5p are a useful tool for influenza diagnosis on influenza A and B.

Multidrug resistance protein 1 (ABCC1) confers resistance to arsenic compounds in human myeloid leukemic HL-60 cells.

Arsenic trioxide (As(2)O(3)) is established as one of the most effective drugs for treatment of patients with acute promyelocytic leukemia, as well as other types of malignant tumors. However, HL-60 cells are resistant to As(2)O(3), and little is known about the underlying resistance mechanism for As(2)O(3) and its biomethylation products, namely, monomethylarsonous acid (MMA(III)) on the treatment of tumors. In the present study, we investigated the molecular mechanisms underlying iAs(III) and its intermediate metabolite MMA(III)-induced anticancer effects in the HL-60 cells. Here, we show that the HL-60 cells exhibit resistance to inorganic iAs(III) (IC(50) = 10 µM), but are relatively sensitive to its intermediate MMA(III) (IC(50) = 3.5 µM). Moreover, we found that the multidrug resistance protein 1 (MRP1), but not MRP2, is expressed in HL-60 cells, which reduced the intracellular arsenic accumulation, and conferred resistance to inorganic iAs(III) and MMA(III). Pretreatment of HL-60 with MK571, an inhibitor of MRP1, significantly increased iAs(III) and MMA(III)-induced cytotoxicity and arsenic accumulations, suggesting that the expression of MRP1/4 may lead to HL-60 cells resistance to trivalent arsenic compounds.

Automated multiplex nucleic acid tests for rapid detection of SARS-CoV-2, influenza A and B infection with direct reverse-transcription quantitative PCR (dirRT-qPCR) assay in a centrifugal microfluidic platform.

The coronavirus disease 2019 (COVID-19) pandemic, caused by the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, has posed a threat to public health worldwide. Also, influenza virus has caused a large number of deaths annually. Since co-infection of SARS-CoV-2 and influenza virus, which share similar symptoms, hampers current treatment efficiency, multiple simultaneous detection of these viruses is needed to provide the right treatment for patients. We developed a microfluidic disc-direct RT-qPCR (dirRT-qPCR) assay for rapid multiplex detection of SARS-CoV-2, influenza A and B viral infection in pharyngeal swab samples in an automated manner. Choices of the DNA polymerase, concentrations of dTPs and MgCl2 were characterized to optimize the assay. A detection limit of 2 × 101 copies per reaction was found in all three viral RNAs with as little as 2 µL of swab samples. The accuracy of our assay was evaluated with 2127 clinical swab samples of infection with these three viruses and healthy controls, and it possessed a consistency rate of 100, 99.54 and 99.25% in SARS-CoV-2, influenza A and B detection in comparison to standard RT-qPCR. The reported scheme of our assay is capable of screening other viral infections for up to 16 targets simultaneously. The whole diagnosis could be completed in 1.5 hours after simple sample loading by a non-technical expert. This constitutes an enabling strategy for large-scale point-of-care screening of multiple viral infections, which ultimately lead to a pathway for resolving the critical issue of early diagnosis for the prevention and control of viral outbreaks.

Technological Advances in Multiscale Analysis of Single Cells in Biomedicine.

Single-cell analysis has recently received significant attention in biomedicine. With the advances in super-resolution microscopy, fluorescence labeling, and nanoscale biosensing, new information may be obtained for the design of cancer diagnosis and therapeutic interventions. The discovery of cellular heterogeneity further stresses the importance of single-cell analysis to improve our understanding of disease mechanism and to develop new strategies for disease treatment. To this end, many studies are exploited at the single-cell level for high throughput, highly parallel, and quantitative analysis. Technically, microfluidics are also designed to facilitate single-cell isolation and enrichment for downstream detection and manipulation in a robust, sensitive, and automated manner. Further achievements are made possible by consolidating optically label-free, electrical, and molecular sensing techniques. Moreover, these technologies are coupled with computing algorithms for high throughput and automated quantitative analysis with a short turnaround time. To reflect on how the technological developments have advanced single-cell analysis, this mini-review is aimed to offer readers an introduction to single-cell analysis with a brief historical development and the recent progresses that have enabled multiscale analysis of single-cells in the last decade. The challenges and future trends are also discussed with the view to inspire forthcoming technical developments.

Development of a direct reverse-transcription quantitative PCR (dirRT-qPCR) assay for clinical Zika diagnosis.

OBJECTIVE: The nucleic acid-based polymerase chain reaction (PCR) assay is commonly applied to detect infection with Zika virus (ZIKV). However, the time- and labor-intensive sample pretreatment required to remove inhibitors that cause false-negative results in clinical samples is impractical for use in resource-limited areas. The aim was to develop a direct reverse-transcription quantitative PCR (dirRT-qPCR) assay for ZIKV diagnosis directly from clinical samples. METHODS: The combination of inhibitor-tolerant polymerases, polymerase enhancers, and dirRT-qPCR conditions was optimized for various clinical samples including blood and serum. Sensitivity was evaluated with standard DNA spiked in simulated samples. Specificity was evaluated using clinical specimens of other infections such as dengue virus and chikungunya virus. RESULTS: High specificity and sensitivity were achieved, and the limit of detection (LOD) of the assay was 9.5×101 ZIKV RNA copies/reaction. The on-site clinical diagnosis of ZIKV required a 5µl sample and the diagnosis could be completed within 2h. CONCLUSIONS: This robust dirRT-qPCR assay shows a high potential for point-of-care diagnosis, and the primer-probe combinations can also be extended for other viral detection. It realizes the goal of large-scale on-site screening for viral infections and could be used for early diagnosis and the prevention and control of viral outbreaks.

Platelet mitochondrial cytochrome c oxidase subunit I variants with benzene poisoning.

BACKGROUND: Chronic benzene poisoning (CBP) is one of the most common chronic occupational poisoning which is associated with mitochondrial oxidative damage, and lead to increasing risk of respiratory diseases such as lung cancer. Cytochrome c oxidase subunit I (COI) is one of the key enzymes that plays an important role in oxidative damage regulation by eliminating reactive oxygen species (ROS). This study investigated the relationship between COI gene variants and the risk of CBP. METHODS: We investigated 44 non-smoking patients who were diagnosed with CBP and 57 unexposed non-smoking controls between the ages of 23 and 60 with their background including work experience, lifestyle and medical records. Peripheral blood (2 mL) was collected in EDTA tube and the platelet was purified from the collected blood. Variants of COI were analyzed by PCR and sequencing. Multivariable linear regression analysis was used to assess the association between CBP exposure and variants. RESULTS: The frequency of the mitochondrial DNA (mtDNA) T6392C, G6962 variants were 10, 7 out of 44 CBP group patients, which was higher when compared to that of 4, 2 out of 57 in the control group, suggesting these variants could be the risk factor for CBP [odds ratio (OR) 3.897, 95% CI: 1.131-13.425, P=0.023; OR 5.203, 95% CI: 1.024-26.442, P=0.034]. There was a significant difference (P<0.05) of COI variants, including T6392C and G6962A, in platelet mtDNA between patients and control samples. Meanwhile, the frequency of the mtDNA C7196A variant were 13 out of 44 control group, which was higher when compared to that of 2 of 57 in the CBP group patients, suggesting this variant could be the protective factor for CBP (OR 6.205, 95% CI: 1.320-29.162, P=0.010). CONCLUSIONS: Our study suggests that T6392C, G6962A and C7196A from platelet mtDNA variants play a significant role in the etiology of CBP and facilitate the development of molecular biomarker on CBP diagnosis.

An Assay Using Localized Surface Plasmon Resonance and Gold Nanorods Functionalized with Aptamers to Sense the Cytochrome-c Released from Apoptotic Cancer Cells for Anti-Cancer Drug Effect Determination.

To determine the degree of cancer cell killing after treatment with chemotherapeutic drugs, we have developed a sensitive platform using localized surface plasmon resonance (LSPR) and aptamers to detect the extracellular cytochrome-c (cyto-c), a mitochondrial protein released from cancer cells for the induction of apoptosis after treatment, to evaluate the effectiveness of cancer therapy. In this assay, a short single-stranded 76-mer DNA aptamer with a unique DNA sequence, which binds towards the cyto-c like an antibody with a high binding affinity and specificity, was conjugated to gold nanorods (AuNR) for LSPR sensing. Practically, cyto-c was first grabbed by a capturing antibody functionalized on the surface of micro-magnetic particles (MMPs). Subsequently, the AuNR-conjugated aptamer was added to form a complex sandwich structure with cyto-c (i.e., (MMP-Ab)-(cyto-c)-(AuNR-aptamer)) after washing away the non-target impurities, such as serum residues and intracellular contents, in a microfluidic chip. The sandwich complex led to formation of AuNR aggregates, which changed the LSPR signals in relation to the amount of cyto-c. With the LSPR signal enhancement effects from the AuNRs, the detection limit of cyto-c, sparked in human serum or culture medium, was found to be 0.1 ng/mL in our platform and the whole sensing process could be completed within two hours. Moreover, we have applied this assay to monitor the apoptosis in leukemia cancer cells induced by a potential anti-cancer agent phenylarsine oxide.