Ultrafast multiplexed detection of SARS-CoV-2 RNA using a rapid droplet digital PCR system.

We report the first combination of droplet digital and rapid PCR techniques for efficient, accurate, and quantitative detection of SARS-CoV-2 RNA. The presented rapid digital PCR system simultaneously detects two specific targets (ORF1ab and N genes) and one reference gene (RNase P) with a single PCR thermal cycling period around 7 s and the total running time less than 5 min. A clear positive signal could be identified within 115 s via the rapid digital RT-PCR, suggesting its efficiency for the end-point detection. In addition, benchmark tests with serial diluted reference samples of SARS-CoV-2 RNA reveal the excellent accuracy of our system (R2>0.99). More importantly, the rapid digital PCR system gives consistent and accurate detection of low-concentration reference samples, whereas qPCR yields Ct values with significant variations that could lead to false-negative results. Finally, we apply the rapid digital PCR system to analyze clinical samples with both positive and control cases, where results are consistent with qPCR test outcomes. By providing similar accuracy with qPCR while minimizing the detection time-consuming and the false-negative tendency, the presented rapid digital PCR system represents a promising improvement on the rapid diagnosis of COVID-19.

Absolute quantification and analysis of extracellular vesicle lncRNAs from the peripheral blood of patients with lung cancer based on multi-colour fluorescence chip-based digital PCR.

Emerging evidence indicates that extracellular vesicle (EV) long non-coding ribonucleic acids (lncRNAs) in lung cancer may be clinically useful biomarkers for early diagnosis using liquid biopsy. However, the extremely low quantities of EV-lncRNAs in peripheral blood are a major challenge for multi-target detection. In this study, we developed a new multi-colour fluorescence digital PCR EV-lncRNA (miDER) analysis chip, and then demonstrated its ability to quickly and accurately analyse the levels of two target genes and one reference gene from peripheral blood. Under the miDER assay, the limit of detection of the target gene from peripheral blood was 10 copies/µL. Based on multiplex assay, the expression levels of two lung cancer-related genes (SLC9A3-AS1 and PCAT6) in patients with lung cancer (n = 32) and healthy controls (n = 30) showed a significant difference between the two groups (P < 0.001; two-tailed t-test). A receiver operating characteristic (ROC) curve analysis was used to evaluate the discrimination ability of these lncRNAs. The combination of two lncRNAs in the miDER assay yielded a higher area under curve (AUC) value of 0.811 (95% CI = 0.705-0.918). Moreover, to determine the absolute quantitation capacity of the miDER assay, we compared the results to those obtained by quantitative real-time polymerase chain reaction (qPCR), demonstrating that the miDER assay is more sensitive than qPCR. The multiplex assay based on the miDER could provide a new solution for the multi-index combined detection of trace EV-lncRNAs in body fluids and demonstrate the use of EV-lncRNAs as biomarkers for lung tumour biopsy.

A microfluidic platform for high-purity separating circulating tumor cells at the single-cell level.

Circulating tumor cells (CTCs) are rare cancer cells that are shed from the tumors into the peripheral blood and are instrumental in distant metastasis. Early detection of CTCs can therefore improve prognoses and help design patient-specific treatment regimen. However, the current CTC isolation techniques have poor efficacy and selectivity, owing to the rarity and heterogeneity of the CTCs. We designed a microchip for integrated single-cell isolation of CTCs - based on cell size and immuno-phenotype - and analysis. Each isolation unit consisted of a trap channel, a bypass channel, and a release channel. The larger cells were preferentially captured at the trap channels and flushed out selectively via release microvalves according to their immuno-phenotype. The average recovery rate and purity of lung cancer cells isolated from a spiked WBC population were respectively 92.5% and 94% using the microchip, which were significantly higher compared to that obtained using anti-CD45 magnetic beads. In addition, the isolated cancer cells were analyzed on chip for the surface markers of epithelial mesenchymal transition. Taken together, the integrated microchip is a promising tool for the isolation and analysis of CTCs in the clinical setting.

Rapid Isolation and Multiplexed Detection of Exosome Tumor Markers Via Queued Beads Combined with Quantum Dots in a Microarray.

Tumor-derived exosomes are actively involved in cancer progression and metastasis and have emerged as a promising marker for cancer diagnosis in liquid biopsy. Because of their nanoscale size, complex biogenesis, and methodological limitations related to exosome isolation and detection, advancements in their analysis remain slow. Microfluidic technology offers a better analytic approach compared with conventional methods. Here, we developed a bead-based microarray for exosome isolation and multiplexed tumor marker detection. Using this method, exosomes are isolated by binding to antibodies on the bead surface, and tumor markers on the exosomes are detected through quantum dot (QD) probes. The beads are then uniformly trapped and queued among micropillars in the chip. This design benefits fluorescence observation by dispersing the signals into every single bead, thereby avoiding optical interference and enabling more accurate test results. We analyzed exosomes in the cell culture supernatant of lung cancer and endothelial cell lines, and different lung cancer markers labeled with three QD probes were used to conduct multiplexed detection of exosome surface protein markers. Lung cancer-derived samples showed much higher (~ sixfold-tenfold) fluorescence intensity than endothelial cell samples, and different types of lung cancer samples showed distinctive marker expression levels. Additionally, using the chip to detect clinical plasma samples from cancer patients showed good diagnostic power and revealed a well consistency with conventional tests for serological markers. These results provide insight into a promising method for exosome tumor marker detection and early-stage cancer diagnosis.

Quantitative assessment of gene promoter methylation in non-small cell lung cancer using methylation-sensitive high-resolution melting.

DNA methylation is closely associated with aberrant epigenetic changes. Previous studies have identified various genes associated with non-small cell lung cancer (NSCLC), but the precise combination responsible for its etiology is still debated. The aim of the present study was to select a new set of NSCLC-related genes using methylation-sensitive high-resolution melting. The promoter methylation status of six selected genes, consisting of protocadherin γ subfamily B, 6 (PCDHGB6), homeobox A9 (HOXA9), O6-methylguanine-DNA methyltransferase (MGMT), microRNA (miR)-126, suppressor of cytokine signaling 3 (SOCS3) and Ras association domain family member 5, also termed NORE1A, was evaluated in 54 NSCLC patients. From these samples, genome-wide DNA was extracted and bisulfite conversion was performed along with fluorogenic quantitative polymerase chain reaction to detect methylation values of the six selected promoters. The present results revealed frequent methylation on PCDHGB6, HOXA9 and miR-126, which contrasted with infrequent methylation on MGMT. The results indicated no methylation on either SOCS3 or NORE1A. The sensitivity and specificity of the methylation assessment were 85.2 and 81.5%, respectively, and the analysis results were validated by pyrosequencing. Furthermore, minute comparison of the association between DNA methylation and clinical features was performed. Overall, these results may provide potential information for the development of better clinical diagnostics and more targeted and effective therapies for NSCLC.

Absolute quantification of DNA methylation using microfluidic chip-based digital PCR.

Hypermethylation of CpG islands in the promoter region of many tumor suppressor genes downregulates their expression and in a result promotes tumorigenesis. Therefore, detection of DNA methylation status is a convenient diagnostic tool for cancer detection. Here, we reported a novel method for the integrative detection of methylation by the microfluidic chip-based digital PCR. This method relies on methylation-sensitive restriction enzyme HpaII, which cleaves the unmethylated DNA strands while keeping the methylated ones intact. After HpaII treatment, the DNA methylation level is determined quantitatively by the microfluidic chip-based digital PCR with the lower limit of detection equal to 0.52%. To validate the applicability of this method, promoter methylation of two tumor suppressor genes (PCDHGB6 and HOXA9) was tested in 10 samples of early stage lung adenocarcinoma and their adjacent non-tumorous tissues. The consistency was observed in the analysis of these samples using our method and a conventional bisulfite pyrosequencing. Combining high sensitivity and low cost, the microfluidic chip-based digital PCR method might provide a promising alternative for the detection of DNA methylation and early diagnosis of epigenetics-related diseases.

Identification of long noncoding RNAs for the detection of early stage lung squamous cell carcinoma by microarray analysis.

The aberrant expressions of long noncoding RNAs have been reported in numerous cancers, which have facilitated the cancer diagnosis. However, the expression profile of lncRNAs in early stage lung squamous cell carcinoma has not been well discussed. The present study aimed to examine the expression profile of lncRNAs in early stage lung squamous cell carcinoma and identify lncRNA biomarkers for diagnosis. Through high-throughput lncRNA microarray, we screened thousands of aberrantly expressed lncRNAs and mRNAs in early stage lung squamous cell carcinoma tissues compared to their corresponding adjacent nontumorous tissues. Bioinformatics analyses were used to investigate the functions of aberrantly expressed mRNAs and their associated lncRNAs. After that, in order to identify lncRNA biomarkers for early detection, candidate lncRNA biomarkers were selected based on our established filtering pipeline and validated by real-time quantitative polymerase chain reaction on a total of 63 pairs of tumor samples. Five lncRNAs were finally identified which were able to distinguish early stage tumor and normal samples with high sensitivity (92%) and specificity (83%). These results imply that lncRNAs may be powerful biomarker for early diagnosis.

Chip-based visual detection of microRNA using DNA-functionalized gold nanoparticles.

In the present study, we developed a highly sensitive and convenient biosensor consisting of gold nanoparticle (AuNP) probes and a gene chip to detect microRNAs (miRNAs). Specific oligonucleotides were attached to the glass surface as capture probes for the target miRNAs, which were then detected via hybridization to the AuNP probes. The signal was amplified via the reduction of HAuCl4 by H2O2. The use of a single AuNP probe detected 10 pmol L(-1) of target miRNA. The recovery rate for miR-126 from fetal bovine serum was 81.5%-109.1%. The biosensor detection of miR-126 in total RNA extracted from lung cancer tissues was consistent with the quantitative PCR (qPCR) results. The use of two AuNP probes further improved the detection sensitivity such that even 1 fmol L(-1) of target miR-125a-5p was detectable. This assay takes less than 1 h to complete and the results can be observed by the naked eye. The platform simultaneously detected lung cancer related miR-126 and miR-125a-5p. Therefore, this low cost, rapid, and convenient technology could be used for ultrasensitive and robust visual miRNA detection.

Absolute quantification of lung cancer related microRNA by droplet digital PCR.

Digital polymerase chain reaction (digital PCR) enables the absolute quantification of nucleic acids through the counting of single molecules, thus eliminating the need for standard curves or endogenous controls. In this study, we developed a droplet digital PCR (ddPCR) system based on an oil saturated PDMS (OSP) microfluidic chip platform for quantification of lung cancer related microRNA (miRNA). The OSP chip was made with PDMS and was oil saturated to constrain oil swallow and maintain the stability of droplets. Two inlets were designed for oil and sample injection with a syringe pump at the outlet. Highly uniform monodisperse water-in-oil emulsion droplets to be used for subsequent detection and analysis were generated at the cross section of the channel. We compared miRNA quantification by the ddPCR system and quantitative real-time PCR (qPCR) to demonstrate that the ddPCR system was superior to qPCR both in its detection limit and smaller fold changes measurement. This droplet PCR system provides new possibilities for highly sensitive and efficient detection of cancer-related genes.

Identification of biomarkers for the detection of early stage lung adenocarcinoma by microarray profiling of long noncoding RNAs.

BACKGROUND: Lung adenocarcinoma has one of the poorest outcomes of any cancer worldwide, in part due to the lack of a reliable means of early detection. Long noncoding RNAs (lncRNAs) have been shown to be deregulated in some types of cancer; however, the contributions of lncRNAs to lung adenocarcinoma remain unknown. METHODS: We described the expression profile of lncRNAs in human lung adenocarcinoma at an early stage and the corresponding adjacent nontumorous tissues (NT) by microarray. From the microarray analysis, a total of 1170 lncRNAs were significantly differentially expressed in three early stage lung adenocarcinoma tissues compared with NT (fold-change≥2.0, p≤0.05). Candidate biomarkers were selected from the significantly differentially expressed lncRNAs based on our established filtering pipeline; subsequently, marker optimization and validation by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) on a total of 102 pairs of early stage lung adenocarcinoma and NT samples. RESULTS: A panel of 5-lncRNA was identified that could distinguish early stage lung adenocarcinoma from NT samples with high sensitivity and specificity. The area under the receiver operating characteristic curve (AUC) for tumor identification in the training and validation sets were 0.978 and 0.987, respectively. CONCLUSIONS: Our results are the first to reveal differentially expressed lncRNAs in early stage lung adenocarcinoma and suggest that lncRNAs may be novel candidate biomarkers for the early detection of this disease.

Early Detection of Lung Cancer in Serum by a Panel of MicroRNA Biomarkers.

INTRODUCTION: The objective of the study was to develop a panel of microRNAs (miRNAs) as highly sensitive and specific biomarkers for lung cancer early detection. MATERIALS AND METHODS: The study contained 2 phases: first, preliminary marker selection based on previous reports on the serum of 24 early stage lung cancer patients and 24 healthy control subjects by TaqMan probe-based real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR); and second, validation of miRNA markers on 94 early stage lung cancer, 48 stage III to IV lung cancer, and 111 healthy control serum samples. RESULTS: A total of 3 miRNAs (miR-125a-5p, miR-25, and miR-126) were selected for further analysis in this study. The combination of the 3 miRNAs could produce 0.936 area under the receiver operating characteristic curve value in distinguishing early stage lung cancer patients from control subjects with 87.5% sensitivity and 87.5% specificity, respectively. The diagnostic value of the miRNA panel in an independent set of lung cancer patients confirmed the sensitivity and specificity. CONCLUSION: The results demonstrated that the panel of miRNA biomarkers had the potential for the early detection of lung cancer.

Multi-nanomaterial electrochemical biosensor based on label-free graphene for detecting cancer biomarkers.

Developing a rapid, accurate and sensitive electrochemical biosensor for detecting cancer biomarkers is important for early detection and diagnosis. This work reports an electrochemical biosensor based on a graphene (GR) platform which is made by CVD, combined with magnetic beads (MBs) and enzyme-labeled antibody-gold nanoparticle bioconjugate. MBs coated with capture antibodies (Ab1) were attached to GR sheets by an external magnetic field, to avoid reducing the conductivity of graphene. Sensitivity was also enhanced by modifying the gold nanoparticles (AuNPs) with horseradish peroxidase (HRP) and the detection antibody (Ab2), to form the conjugate Ab2-AuNPs-HRP. Electron transport between the electrode and analyte target was accelerated by the multi-nanomaterial, and the limit of detection (LOD) for carcinoembryonic antigen (CEA) reached 5 ng mL(-1). The multi-nanomaterial electrode GR/MBs-Ab1/CEA/Ab2-AuNPs-HRP can be used to detect biomolecules such as CEA. The EC biosensor is sensitive and specific, and has potential in the detection of disease markers.

Rolling circle amplification immunoassay combined with gold nanoparticle aggregates for colorimetric detection of protein.

A highly sensitive and novel colorimetric rolling circle amplification (RCA) immunoassay for detecting C-reactive protein (CRP) has been developed. In the assay, a CRP capture antibody was immobilized on magnetic beads and a CRP detection antibody was conjugated with single-stranded DNA (ssDNA) using N-[ε-maleimidocaproyloxy] sulfosuccinimide ester. Along with the addition of CRP, a "sandwich" structure was formed. Subsequently, the ssDNA was used as a primer to initiate the RCA reaction in the presence of the circular template, phi29 DNA polymerase and deoxynucleotide triphosphates. The RCA product obtained by magnetic separation, and long tandem repeated sequences mediated the aggregation of gold nanoparticles (AuNPs), which could be observed by the naked eye or quantified using absorption spectra with a detection limit of 30 fg mL(-1) and a linear response range from 10 ng mL(-1) to 1 pg mL(-1). This assay offers the advantages of isothermal conditions, low cost and label-free quantification that could be hopeful for ultrasensitive and robust visual protein detection.