Microchip Analytical Procedures Research Articles

POCT in diagnosis of infectious diseases—the state of art and future

The pathogen diagnosis of infectious diseases plays a critical role in the rational application of antibiotics, the monitoring and management of disease epidemiology and the control of drug-resistant bacteria in hospitals and communities. The ideal method for rapid detection of infectious diseases should be sensitive, rapid, specific, stable, low-cost and easy to use. This article reviews the clinical application of POCT in infectious disease antigen detection, antibody detection, nucleic acid detection, etc. and focuses on the advantages and potential of microfluidic technology in pathogen multiplex nucleic acid detection and drug resistance detection. Choosing the right time, the right place, and the right on-site rapid diagnostic technology will provide a new strategy for the diagnosis and treatment of infectious diseases in the graded diagnosis and treatment mode. (Chin J Lab Med, 2018, 41: 641-643) Key words: Infections diseases; Point-of-care testing; Microfluidics; Microchip analytical procedures

The application of microfluidic systems in clinical diagnostics

Currently, clinical laboratories are developing towards two different directions. One is centralized laboratory with automated pipelines, and the other is highly integrated portable detection system.The latter evolved from an early single test device gradually to an integrated platform based on microfluidic technology, which can operate multiple steps and tests simultaneously. The microfluidic system is further developed to a miniaturized device which can perform biochemical, immune, microbial and cytological tests. With the medical reform process in China, the microfluidic systems will appear in the primary health unit or even families. This review concludes the current application of microfluidic technology in clinical diagnostics and its future trends.(Chin J Lab Med, 2018, 41: 631-633) Key words: Microchip analytical procedures; Microfluidics; Clinical laboratory techniques

LncRNAs expression profile of ankylosing sporidylitis revealed by microarray

Objective To investigate the expression profile variation of long non-coding RNAs (lncRNAs) in ankylosing sporidylitis (AS) and explore the role of lncRNAs in the pathogenesis of AS. Methods The peripheral blood mononuclear cells of AS patients and health controls (HC) were used to detect for differently expressed lncRNAs by microarray. The roles of lncRNAs were predicted with GO and pathway analysis. The results were verified by real time-polymerase chain reaction (PCR). Results A total of 148 lncRNAs and 134 mRNAs were detected, which had more than 2-fold differentially expressed in AS patients. Bioinformatics analysis found that GO term enrichment included protein binding, regulation of transcription, metabolism, signal transduction, et al. and might involve in toll-like receptor pathway, protein kinase, complement pathway, notch signaling pathway and so on. The expressions of three lncRNAs were estimated by real time-PCR which found that consistent with that of microarrays. Among these, D90064 was the most aberrantly expressed lncRNAs. Conclusions Several lncRNAs expression was changed significantly in AS patients in comparison with HC, which implies that those different lncRNAs may have an important role in the development and progression of AS. Key words: RNA/ME; Spondylitis, ankylosing/ME; Microchip analytical procedures

MicroRNA expression profiles in peripheral blood and skin lesions of patients with psoriasis vulgaris

Objective To investigate microRNA (miRNA) expression profiles in peripheral blood and skin lesions of patients with psoriasis vulgaris (PV) , and to seek miRNAs consistently expressed in peripheral blood and skin lesions. Methods A miRNA microarray was used to screen differentially expressed miRNAs in skin lesions and peripheral blood samples between 17 patients with PV and 4 healthy human controls, and real-time fluorescence-based quantitative PCR (RT-qPCR) to verify the differentially expressed miRNAs. The correlations of these differentially expressed miRNAs with psoriasis area and severity index (PASI) score were assessed by Pearson correlation analysis. Results The Agilent human miRNA microarray profiling revealed 15 miRNAs consistently expressed in skin lesions and peripheral blood of patients with PV. Of the 15 miRNAs, 7 were verified as consistently expressed miRNAs by RT-qPCR. Among the 7 miRNAs, the expression intensity of miR-30e-5p, miR-192-5p, miR-17-3p and miR-1227-5p was negatively correlated with PASI scores (all P 0.05) . Conclusion Some miRNAs are consistently expressed in skin lesions and plasma of PV patients, which are expected to serve as biomarkers for evaluation of psoriasis severity. Key words: Psoriasis; MicroRNAs; Microchip analytical procedures; Gene expression profiling; Severity of illness index

Label-free imaging for living cell detection

The living cell imaging technique can observe the internal structure and physiological processes of living cells in real time or a period of time, which can provide more reliable information compared with the fixed cell imaging method. Most of the existing techniques for living cell imaging are based on fluorescence imaging technology, which through increasing brightness of the fluorescent groups to reduce cytotoxicity and obtain high signal to noise ratio of the image. These kinds of methods will cause certain influence on the activity of the cells. The improvement of modern physical technology makes label-free imaging method for living cell detection become possible. This paper reviews the current state of label-free imaging methods for living cell detection which may provide a new method for living cell detection.(Chin J Lab Med, 2016, 39: 801-803) Key words: Cytological techniques; Biosensing techniques; Microchip analytical procedures; Spectrum analysis, raman; Terahertz imaging

Differential expression of microRNA in paraffin-embedded tissue in patients with multiple and solitary gliomas: a preliminary study

Objectives To compare the differences of microRNA (miRNA) expression profile of multiple and solitary gliomas in formalin-fixed and paraffin embedded (FFPE) tissue and to preliminarily investigate the possible roles in glioma invasion and migration for the significantly different miRNA and its target genes. Methods FFPE specimens in patients with multiple glioma (n=6) and solitary glioma (n=6) were selected. The total RNA was extracted and detected; the gene chip technology was used to detect miRNA expression profile. The chip analysis software was used to analyze the data of the results. The miRNAs of significant expression differences of the multiple and solitary gliomas were screened. Then the biological information database was used to predict the target genes of miRNA of the most significant expression differences. Results The total RNA quality extracted from all the FFPE tissue specimens met the standard of quality control. There were significant differences in 128 different miRNAs in the multiple glioma compared with the solitary glioma (multiple >5 or 50), they were miR-21, miR-455, miR-199, miR-410, miR-148a, miR-335, miR-34b, miR-664a, miR-542, and miR-4453, respectively. Five miRNAs were significantly downregulated (multiple <0.05), they were miR-200a, miR-451, miR-29a, miR-101, and miR-96. Based on the most significant prediction results of the miR-21 and miR-200a target genes of upregulation and downregulation, their multiple target genes involved in tumor invasion and migration related biological function. Conclusions FFPE tissue may meet the subsequent experimental study, such as miRNA chip, etc. and thus provide rich resources for the research of the miRNA biological function and exploration. Multiple glioma has differential expression of miRNA compared with solitary glioma, in which miR-21and miR-200a may be closely associated with the invasion and migration of glioma. Key words: Glioma; MicroRNAs; Microchip analytical procedures; Neoplasm invasiveness; Cell migration

Transcriptomics of psoriasis

Psoriasis is a chronic inflammatory multigenic dermatosis of unclear etiology. Recently, more and more studies have been carried out to investigate into its physiopathologic mechanism at transcriptome level. The methods used in these studies mainly include expressed sequence tag(EST)analysis, GeneChip, second-generation sequencing technology, etc. Some non-coding RNAs, including miR-21, miR-203 and miR-31, have been found to be expressed abnormally in psoriatic lesions, and closely related to the differentiation and proliferation of keratinocytes. These findings provide important evidences for the pathogenesis of psoriasis. Key words: Psoriasis; Microchip analytical procedures; Multilocus sequence typing

Establishment of a novel mutant-enriched liquid chip technology for detecting EGFR mutations in plasma of patients with non-small cell lung cancer

Objective To establish a sensitive, specific, simple and high-throughput method for detection of the epidermal growth factor receptor (EGFR) mutation in the plasma samples of patients with non-small cell lung cancer (NSCLS) by the use of mutant-enriched liquid chip (MEL) assay. Methods The specific probes for the EGFR exon19 E746-750 deletion, exon 21 L858R mutation and wild-type sequence were designed and coupled to the microspheres coding with different fluorescent dye.The probe coupling efficiency was verified by crossing hybridization test with biotin-labeled reverse sequence.A blood-based MEL approach which integrates a sensitive mutant-enriched PCR and quantitative high throughput liquid chip assay for assessment of EGFR mutations was developed.The sensitivity and specificity of MEL was further evaluated using the mixture with different copy numbers of mutant and wild-type plasmids as template.The mutations of exon 19 and 21 of EGFR gene in plasma samples from 201 patients with stage ⅢB or Ⅳ NSCLC who enrolled in the First Affiliated Hospital of Guangzhou Medical College from September 2008 to April 2010 were analyzed by both the MEL and the mutant-enriched PCR assay.The result comparison was made between direct sequencing and MEL in 50 cases whose EGFR gene type had been tested by MEL and mutant-enriched PCR.The correlation of EGFR gene mutation and the response to the Geftinib treatment was analyzed in 16 patients with lung adenocarcinoma as well. Results The probes were successfully coupled to the microspheres encoding with different fluorescent dye, and could be specifically recognized by the corresponding target sequence.The MEL was capable of detecting as few as 10 copies of EGFR mutants (sensitivity was 0.1%).Among the enrolled 201 cases of advanced NSCLC, the detection rate of the EGFR exon19 E746-750 del and exon 21 L858R was 55.7% (112/201) by MEL assay.Compared with mutant-enriched PCR［58.2%（117/201）］, the coincidence rate was 97.5%(196/201).There was no statistically significant difference between the results of mutant-enriched PCR and MEL (χ2=3.20，P>0.05).The mutations detection rate was 22.0% (11/50) by directing sequencing, which was significantly lower than by MEL［50.0%(25/50), χ2=12.07，P<0.05］.Among the 16 patients treated with Gefitinib，9 cases who had EGFR mutation showed a higher response rate（P=0.041）and prolonged progression-free survival（χ2=6.76，P=0.009）after the treatment compared to those 7 who without EGFR mutation. Conclusions A new method of MEL with accuracy, specificity, fast and high-throughput is established for the detection of EGFR 19 E746-750 deletion and exon 21 L858R mutations in plasma from advanced NSCLC patients.It has the ability to provide the most direct and valuable guidance for clinicians to make decision on EGFR tyrosine kinase inhibitors therapy in the advanced NSCLC patients.(Chin J Lab Med, 2012,35:986-992) Key words: Carcinoma; non-small-cell lung; Receptor; epidermal growth factor; Mutation; Microchip analytical procedures

The significance and the change of miRNAs expression profile of peripheral blood mononuclear cell in different period of HBV infection

Objective To explore the significance and the change of miRNAs expression profile in different period of HBV infection. Methods Establish the detection method of microRNA(miRNA) by RNA DNA probe liquid chip (flexible multi-analyte profiling, xMap) and estimate the specificity, repeatability and accuracy of this method.From October 2010 to October 2011, collect HBV infected patients′ peripheral blood of the First affiliated hospital of SooChow University, including acute hepatitis B, chronic hepatitis B, hepatits B liver cirrhosis, liver cancer patients and health control,each group contains 40 cases.The levels of miR-191,-223,-222,-145,-21,-31,-126,-20a,-372 of peripheral blood mononuclear cell were detected by xMap liquid chiptechnology.The level of miR-103 was taken as reference.The ratio of (the mean fluorescence intensity of target miRNAs-corresponding backgroud mean fluorescence intensity)/(the mean fluorescence intensity of miR-103-corresponding backgroud mean fluorescence intensity) as a valid data and analysis the characteristics of miRNAs expression.The SPSS 17.0 was used as statistical software.The single factor analysis of variance was used as the method to analysis group comparison,and make multiple comparison by the LSD-t method if the result with a significant difference. Results The specificity of xMAP liquid chip method to detect miRNAs was 100%;The repeated experiment proved that the CV value was less than 5% in the high value reference miRNA test, less than 10% in the low value reference miRNAs test; the accruracy experiment proved that the recovery rate was (100±5)% in the nine miRNAs.There were no statistically differences with miR-222(F=1.32, P>0.05）,-191（F=1.98,P>0.05）,-145（F=0.78, P>0.05）,- 21（F=0.64,P>0.05）,-31（F=0.83,P>0.05）,-372（F=1.75,P>0.05）in different groups; There was statistically significant differences in miR-223（F=14.56，P<0.05）among different groups, with the highest expression level in actue hepatitis B group （15.37±4.01）,and the lowest expression level in liver cancer group (6.91±3.18); There was statistically significant differences in miR-126 （F=17.43,P<0.05）among different groups,with the highest expression level in health control group （6.33±2.75） and the lowest expression level in liver cancer group （2.38±1.07）.There were statistically significant differences in miR-20a（F=19.484，P<0.05） among different groups,with the highest expression level in health control group (0.33±0.18) and the lowest expression level in liver cancer group (0.81±0.24). Conclusion The detection method of miRNA by xMAP liquid chip has strong specificity, high accuracy and good repeatability, suitable for large throughput clinical testing.The study for miRNA in HBV infected diseases provides a new clue to the research of chronic progress mechanism.（Chin J Lab Med，2012,35：544-549） Key words: Hepatiti B virus; Leukocytes; mononuclear; microRNAs; Hepatitis B; Microchip analytical procedures

The expression of arginase I in heptocellular carcinoma and its clinicopathological significance

Objective To investigate the expression of arginase Ⅰ(ARG1)in hepatocellular carcinoma(HCC)and to analyze its correlation with clinicopathological features.Methods The expression of ARG1 at protein level in 167 samples of HCC and corresponding adjacent liver tissue was detected with high-throughput tissue microarray technique and immunohistochemistry.The correlation between ARG1 expression and clinicopathological features was analyzed with x2 test and Spearman rank correlation analysis.The expression of ARG1 at mRNA level in 68 samples of HCC and corresponding adjacent liver tissue was determined by real-time polymerase chain reaction(real-time PCR).Results The expression of ARG1 at protein level in HCC(3.540±3.702)was significantly lower than that of the corresponding adjacent liver tissues(10.290 ± 2.303)(t=-22.421,P=0.000).The ARG1 expression was correlated with differentiation degree of HCC,histological grade,vascular invasion,preoperative level of α-fetoprotein(AFP)and recurrence after operation(all P＜0.05).The ARG1 expression at mRNA level in 68 HCC tissue[0.0997(0.213)]was lower than that of the corresponding adjacent liver tissues[0.563(0.459)],and the difference was statistical significant(u=-6.544,P=0.000).Conclusion Low expression of ARG1 in HCC may take part inHCC genesis and development.Detecting the expression of ARG1 may be helpful in HCC diagnosis,differentiation degree and prognosis assessment. Key words: Carcinoma, Hepatocellular; Arginase; Polymerase chain reaction; Microchip analytical procedures; Immunohistochemistry; Prognosis

Rapid detection of clinical common bacteria using DNA microarray

Objective To detect eight kinds of clinical common pathogenic bacteria by DNA microarray.Methods Eight kinds of common pathogenic bacteria,including Staphylococcus aureus,Pseudomonas aeruginosa,Klebsiella pneumoniae,Escherichia coli,Proteus mirabilis,Enterobacter aerogenes,Pseudomonas fluorescens,Shigella sonnei were collected.Universal primers were designed to amplify 16S rRNA gene fragment from the genomic DNA of the eight bacteria,and probes were designed in the highly variable regions.DNA microarray detection system was established and used for detection of colleted bacteria.A total of 50 samples were collected from the Zhongnan Hospital of Wuhan University,including 6 blood samples,32 sputum samples,9 feces samples and 3 bronchoscope lavage samples.DNA were extracted and detected by the established DNA microarray system.Results The desired fragments were well amplified by the self-designed universal primers.The selected probes had good detection results according to repeated detection.Of the 50 samples detected,pathgenic bacteria were accurately detected in 47 samples.Other three samples were not detected as those bacteria were not included in the chip.By optimizing the detection process,the results could be reported within 8 hours.Observation of probe signal attenuation indicated that even attenuated after 60 days,but the attenuation did not affect the results.Conclusion A microarray system was established for detection of clinical common bacteria accurately and quickly,which provided foundation for its clinical application. Key words: Microchip analytical procedures; Bacteriological techniques; RNA,ribosomal, 16S

Biochips:valuable vehicles for translational medicine

Biochips have become important analytical tools in biomedical research and clinical diagnostics because they have the advantage to integrate many different analytical steps, such as sample preparation, chemical reactions, analytical separation, all on the same device. There are various types of biochips including microfluidic chips, microarray chips and bioelectronic chips. This article describes the progress of biochip technology, clinical applications and limitations, and technical outlook. Key words: Microchip analytical procedures; Laboratory techniques and procedures

Initial microarray analysis on different fractionated radiation regimens in xenografts with human lung adenocarcinoma

Objective To indentify the gene expression on different fractionated radiation regimens with the same total radiation dose in xenografts with human lung adenocareinoma. Methods Forty-eight BALB/c-nu mice, implanted with human lung adenocarcinoma (Anip973), were randomized into 4 groups: normal control greup,60 Gy in 30 fractions conventional radiation group (2 Gy group) ,60 Gy in 10 fractions hypofractionated radiation group (6 Gy group) ,60 Gy in 6 fractions hypofractionaed radiation group (10 Gy group). Gene alterations were investigated with the microchip analytical procedures covering the entire genome. Genes with significantly different expression were further validated by the quantitative real-time polymerase chain reaction (RT-PCR). Results Compared to the 2 Gy group, the expression of the genes related with the cell growth inhibition and apoptesis was increased, while the genes related with the cell proliferation, anti-apoptosis and DNA damage repair were decreased in the 6 Gy and 10 Gy groups. Confirmed by RT-PCR, c-myc gene was distinctly suppressed in the 6 Gy group (2. 9%) comparing with 2 Gy (5.6%) group and 10 Gy (4.8%) group (P=0. 000,P=0. 002) , and was slightly suppressed in the 10 Gy group comparing with 2 Gy group (P = 0. 069). Conclusions In the BALB/c-nu mice implanted with human lung adenocarcinoma, the hypofractionated radiation regimens clearly inhibit the tumor growth more than conventional fractionation group, though with the same total dose. The 6 Gy group seem to be more effective than 10 Gy group in the inhibition of tumor growth. Key words: Cell lines, lung adenocarcinoma; Irradiation, fractionation; Gene expression, microchip analytical procedures

Application of liquid chip-mass spectrometry technology for screening serum biomarker proteins in lung squamous cell carcinoma

To screen the serum biomarker proteins of lung squamous cell carcinoma (SCCs) by liquid chip-mass spectrometry technology. All serum samples, including 34 SCCs, 46 benign lung diseases (BLDs) and 44 healthy individuals, were analyzed by CLINPROT system in order to study the serum protein expression profiles. Then the discriminatory proteins were detected by FlexAnalysis 3.0 software. Biomarkers were identified by liquid chromatography-tandem mass spectrometry (LCMS/MS). Comparing the differential serum expression proteins between SCCs and healthy individuals, and SCCs and BLDs respectively. Ninety-six differential protein peaks [mass-to-charge ration (M/Z) between 800 and 10 000] were found between SCCs and healthy individuals. In these protein peaks, the expression of protein peaks at 4054.13 M/Z and 4267.46 M/Z had the largest difference between them. The two protein peaks could accurately separate SCCs from healthy individuals by the frame of axes. Similarly, 99 differential protein peaks were automatically detected between SCCs and BLDs. In these protein peaks, the expression of protein peaks at 5065.27 M/Z and 4054.02 M/Z had the largest difference between them. The two protein peaks could accurately separate SCCs from BLDs by the frame of axes. Identified by LC-MS/MS, 1778 M/Z and 1865 M/Z might be assayed jointly and corresponded to complements C3 fragment or C3f precursor. Differential protein expressions existed between SCCs versus healthy individuals and SCCs versus BLD patients. It is feasible to screen the diagnostic serum biomarkers of SCC with a high sensitivity and specificity by using CLINPROT system.

Development of a high-throughput suspension microarray technology for detection of three kinds of veterinary drug residues: chloramphenicol, clenbuterol and 17-beta-estradiol

To establish a novel suspension microarray technology for the detection of three kinds of veterinary drug residues: chloramphenicol, clenbuterol and 17-beta-estradiol (CAP, CL and E2). The three conjugates that veterinary drug coupled with bovine serum albumin (BSA) were synthesized and identified by ultraviolet (UV) spectrophotometry and mass spectrum. The veterinary drug conjugates were immobilized on the polystyrene fluorescent microspheres/beads. There were competitive reactions between the veterinary drugs in the aqueous phase and that on the beads for combination with their specific biotinylated monoclonal antibodies. The optimum amount of the veterinary drug conjugates and the antibodies were optimized and selected. The detective standard curves were plotted. The specificity and the unknown samples were also determined by grouping according to different concentrations of the interferes and the samples. Meantime, the different microstructures of the surfaces of the beads were also observed by scanning electron microscope. Couplings were completed between small molecular veterinary drugs and BSA. The amounts of the three conjugates and the antibodies were optimized. The detective standard curves of the suspension array and their corresponding coefficients of determination (R2) were good (R2 > 0.99). The detection ranges of the three veterinary drugs were (40.00 - 6.25) x 10(5) ng/L, (50.00-7.81) x 10(5) ng/L and 1.00 x 10(3) - 7.29 x 10(5) ng/L respectively. Simultaneously, the specific detection of the suspension microarray was excellent and did not indicate significant cross-reactions. Errors between the found and the real are in the range of 8.09% - 17.03%. It can be considered that the relative standard deviations were relatively small. Successful couplings were also directly confirmed by the observation for microstructures of the surfaces of the beads by scanning of electron microscope and laid good foundation for the following responses. The high-throughput suspension microarray should provide a novel method for multi-analysis of the veterinary drugs and have a wide applicative prospects with simple operation, sensitive, rapid and low cost.