Zule Cheng

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.

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.

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.

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.

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.

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.

An emulsion digital PCR quantitative method based on microbeads and micropillar array chip

BEAMing (beads, emulsion, amplification, and magnetics) is a high sensitive emulsion digital PCR (polymerase chain reaction) technology. Target beads obtained via BEAMing can be used to determine the copy number of target DNA population. However, the existing beads counting process requires specialized instrument, which has increased the detection costs. Here we developed an easy and low cost beads counting method combined with microspheres and micropillar array chips. Through counting the chip-trapped polystyrene microspheres which have captured the target magnetic beads, we could easily infer the number of target magnetic beads and the copy number of target DNA. The trapping mechanism was based on the difference of microsphere diameter and pillar intervals. Fabrication of the chip was simple and low-cost. This method has reduced the difficulty of beads counting process in BEAMing and may promote the applications of BEAMing in clinical filed.

Long Non-Coding RNA in Non-Small Cell Lung Cancers

Non-small cell lung cancer (NSCLC) accounts for nearly 80% of diagnosed lung cancers. Due to the predominantly late diagnosis of NSCLC and drug resistance in the targeted therapy approaches, the 5-year overall survival rate is still less than 19%. Thus, novel diagnosis and treatment approaches are needed. Many efforts have been made to achieve great progress in understanding the genomic landscape of NSCLC and the molecular mechanisms involved in tumorigenesis. Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides with little or no protein-coding potential. They are encoded across the genome and are involved in a wide range of cellular and biological processes. Dysregulation of lncRNAs is associated with a number of cancerrelated processes, including epigenetic regulation, microRNA silencing, and DNA damage. Furthermore, lncRNAs have been reported to have the potential as biomarker for diagnosis and prognosis, as well as the therapy targets. Here in this chapter, we review some well-characterized lncRNAs associated with NSCLCs and the potential of lncRNAs as biomarkers in the diagnosis and prognosis of NSCLCs.