Plasmonic Gold Chip for Multiplexed Detection of Ovarian Cancer Biomarker in Urine

Human epididymal protein 4(HE4),carbohydrate antigen 125(CA125)and osteopontin(OPN)are three key biomarkers in detecting ovarian cancer.To explore the diagnosis value of combined detection of these three biomarkers for ovarian cancer,we developed a multiplexed assay on a plasmonic gold(pGOLD)platform for measuring HE4,CA125 and OPN in urine.The receiver operator characteristic(ROC)curve was drawn,and the diagnosis values of each biomarker alone or in combination for ovarian cancer were evaluated.In the analysis to distinguish ovarian cancer from other gynecological cancers,ovarian cysts and healthy people,the sensitivities of HE4,CA125 and OPN were 72.55%,52.82%and 68.63%,the specificity values were 95.06%,87.65%and 90.12%,while the areas under the curve(AUC)were 0.85,0.75 and 0.77,respectively.The sensitivity and specificity for combination detection of the three markers were 90.20%and 80.25%.The detection methods of HE4,CA125 and OPN based on plasma fluorescence enhanced chip showed good analytic and diagnostic performance,and provided a non-invasive method for the diagnosis of ovarian cancer.

Establishment and performance analysis of a new multiplex detection method for influenza an and B virus antigen

BACKGROUND Influenza A and B virus detection is pivotal in epidemiological surveillance and disease management.Rapid and accurate diagnostic techniques are crucial for timely clinical intervention and outbreak prevention.Quantum dot-encoded microspheres have been widely used in immunodetection.The integration of quantum dot-encoded microspheres with flow cytometry is a well-established technique that enables rapid analysis.Thus,establishing a multiplex detection method for influenza A and B virus antigens based on flow cytometry quantum dot microspheres will help in disease diagnosis.AIM To establish a codetection method of influenza A and B virus antigens based on flow cytometry quantum dot-encoded microsphere technology,which forms the foundation for the assays of multiple respiratory virus biomarkers.METHODS Different quantum dot-encoded microspheres were used to couple the monoclonal antibodies against influenza A and B.The known influenza A and B antigens were detected both separately and simultaneously on a flow cytometer,and the detection conditions were optimized to establish the influenza A and B antigen codetection method,which was utilized for their detection in clinical samples.The results were compared with the fluorescence quantitative polymerase chain reaction(PCR)method to validate the clinical performance of this method.RESULTS The limits of detection of this method were 26.1 and 10.7 pg/mL for influenza A and B antigens,respectively,which both ranged from 15.6 to 250000 pg/mL.In the clinical sample evaluation,the proposed method well correlated with the fluorescent quantitative PCR method,with positive,negative,and overall compliance rates of 57.4%,100%,and 71.6%,respectively.CONCLUSION A multiplex assay for quantitative detection of influenza A and B virus antigens has been established,which is characterized by high sensitivity,good specificity,and a wide detection range and is promising for clinical applications.

Establishment of Multiplex PCR for Simultaneous Detection of Four Venereal Pathogens

Venereal diseases are considered to be the most prevalent infectious diseases in the worldwide. China is now faced with a year-by-year increasing incidence of sexually transmitted diseases （STD）, which are spreading from high-risk groups to the general population. Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum and herpes simplex virus-2 （HSV-2） are always regarded as the most common venereal pathogens. The ＂golden standard＂ for testing Neisseria gonorrhoeae remains to be bacteria culture or microscopic examination.

Development of a duplex real-time PCR method for the detection of influenza C and D viruses

Influenza viruses are major respiratory pathogens known to infect human and a variety of animals and are widely prevalent worldwide.Genome structure of influenza D virus(IDV)is identical to that of influenza C virus(ICV),and phylogenetic analyses suggest that IDV and ICV share a common ancestry and high homology.To date,the prevalence of ICV and IDV in China is unclear,but these viruses represent a potential threat to public health due to cross-species transmission and zoonotic potential.To efficiently monitor ICV and IDV,it is necessary to establish a dual detection method to understand their prevalence and conduct in-depth research.A duplex real-time PCR method for the simultaneous detection of ICV and IDV was developed.TaqMan fluorescent probes and specific primers targeting NP gene of ICV and PB1 gene of IDV were designed.This method exhibited good specificity and sensitivity,and the detection limit reached 1 × 10^(1) copies/pL of plasmid standards of each pathogen.Thirty-one clinical swine samples and 10 clinical cattle samples were analyzed using this method.One positive sample of IDV was detected,and the accuracy of clinical test results was verified by conventional PCR and DNA sequencing.The duplex real-time PCR detection method represents a sensitive and specific tool to detect IG/and IDV,It provides technical support for virus research and clinical diagnosis of ICV and IDV.This information will benefit animal and human health.

Multiplexed intracellular detection based on dual-excitation/dualemission upconversion nanoprobes

Multiplexed intracellular detection is desirable in biomedical sciences for its higher eficiency and accuracy compared to the single-analyte detection.However,it is very challenging to construct nanoprobes that possess multiple fluorescent signals to recognize the different intracellular species synchronously.Herein,we proposed a novel dual-excitation/dual-emission upconversion strategy for multiplexed detection through the design of upconversion nanoparticles(UCNP)loaded with two dyes for sensitization and quenching of the upconversion luminescence(UCL),respectively.Based on the two independent energy transfer processes of near-infrared(NIR)dye IR845 to UCNP and UCNP to visible dye PAPS-Zn,CIO-and Zn2+were simultaneously detected with a limit of detection(LOD)of41.4 and 10.5 nM,respectively.By tilizing a purpose built 830/980 nm dual-laser confocal microscope,both intrinsic and exogenous CIO and Zn2+in live MCF-7 cells have been accurately quantified.Such dual-excitation/dual-emission ratiometric UCL detection mode enables not only monitoring multiple intracellular analytes but also eliminating the detection deviation caused by inhomogeneous probe distribution in cells.Through modulation of NIR dye and visible dye with other reactive groups,the nanoprobes can be extended to analyze various intraellular species,which provides a promising tool to study the biological activities in live cells and diagnose diseases.

Using oligonucleotide suspension arrays for laboratory identification of bacteria responsible for bacteremia

The aim of this study was to develop and validate an oligonucleotide suspension array for rapid identification of 15 bacterial species responsible for bacteremia, particularly prevalent in Chinese hospitals. The multiplexed array, based on the QIAGEN LiquiChip Workstation, included 15 oligonucleotide probes which were covalently bound to different bead sets. PCR amplicons of a variable region of the bacterial 23S rRNA genes were hybridized to the bead-bound probes. Thirty-eight strains belonging to 15 species were correctly identified on the basis of their corresponding species-specific hybridization profiles. The results show that the suspension array, in a single assay, can differentiate isolates over a wide range of strains and species, and suggest the potential utility of suspension array system to clinical laboratory diagnosis.

A multiplexed circulating tumor DNA detection platform engineered from 3D-coded interlocked DNA rings

Circulating tumor DNA(ctDNA)is a critical biomarker not only important for the early detection of tumors but also invaluable for personalized treatments.Currently ctDNA detection relies on sequencing.Here,a platform termed three-dimensional-coded interlocked DNA rings(3D-coded ID rings)was created for multiplexed ctDNA identification.The ID rings provide a ctDNA recognition ring that is physically interlocked with a reporter ring.The specific binding of ctDNA to the recognition ring initiates target-responsive cutting via a restriction endonuclease;the cutting then triggers rolling circle amplification on the reporter ring.The signals are further integrated with internal 3D codes for multiplexed readouts.ctDNAs from non-invasive clinical specimens including plasma,feces,and urine were detected and validated at a sensitivity much higher than those obtained through sequencing.This 3D-coded ID ring platform can detect any multiple DNA fragments simultaneously without sequencing.We envision that our platform will facilitate the implementation of future personalized/precision medicine.

Wave-shaped microfluidic chip assisted point-of-care testing for accurate and rapid diagnosis of infections

Background:Early diagnosis and classification of infections increase the cure rate while decreasing complications,which is significant for severe infections,especially for war surgery.However,traditional methods rely on laborious operations and bulky devices.On the other hand,point-of-care(POC)methods suffer from limited robustness and accuracy.Therefore,it is of urgent demand to develop POC devices for rapid and accurate diagnosis of infections to fulfill on-site militarized requirements.Methods:We developed a wave-shaped microfluidic chip(WMC)assisted multiplexed detection platform(WMC-MDP).WMC-MDP reduces detection time and improves repeatability through premixing of the samples and reaction of the reagents.We further combined the detection platform with the streptavidin–biotin(SA-B)amplified system to enhance the sensitivity while using chemiluminescence(CL)intensity as signal readout.We realized simultaneous detection of C-reactive protein(CRP),procalcitonin(PCT),and interleukin-6(IL-6)on the detection platform and evaluated the sensitivity,linear range,selectivity,and repeatability.Finally,we finished detecting 15 samples from volunteers and compared the results with commercial ELISA kits.Results:Detection of CRP,PCT,and IL-6 exhibited good linear relationships between CL intensities and concentrations in the range of 1.25–40μg/ml,0.4–12.8 ng/ml,and 50–1600 pg/ml,respectively.The limit of detection of CRP,PCT,and IL-6 were 0.54μg/ml,0.11 ng/ml,and 16.25 pg/ml,respectively.WMC-MDP is capable of good adequate selectivity and repeatability.The whole detection procedure takes only 22 min that meets the requirements of a POC device.Results of 15 samples from volunteers were consistent with the results detected by commercial ELISA kits.Conclusions:WMC-MDP allows simultaneous,rapid,and sensitive detection of CRP,PCT,and IL-6 with satisfactory selectivity and repeatability,requiring minimal manipulation.However,WMC-MDP takes advantage of being a microfluidic device showing the coefficients of variation less than 10%enabling WMC-MDP to be a type of point-of-care testing(POCT).Therefore,WMC-MDP provides a promising alternative to POCT of multiple biomarkers.We believe the practical application of WMC-MDP in militarized fields will revolutionize infection diagnosis for soldiers.

Enzyme-free and multiplexed micro RNA detection using micro RNA-initiated DNA molecular motor

In this work,we have developed a sensitive,simple,and enzyme-free assay for detection of micro RNAs(mi RNAs)by means of a DNA molecular motor consisting of two stem-loop DNAs with identical stems and complementary loop domains.In the presence of mi RNA target,it can hybridize with one of the stem-loop DNA to open the stem and to produce a mi RNA/DNA hybrid and a single strand(ss)DNA,the ss DNA will in turn hybridize with another stem-loop DNA and finally form a double strand(ds)DNA to release the mi RNA.One of the stem-loop DNA is double-labeled by a fluorophore/quencher pair with efficiently quenched fluorescence.The formation of ds DNA can produced specific fluorescence signal for mi RNA detection.The released mi RNA will continuously initiate the next hybridization of the two stem-loop DNAs to form a cycle-running DNA molecular motor,which results in great fluorescence amplification.With the efficient signal amplification,as low as 1 pmol/L mi RNA target can be detected and a wide dynamic range from 1 pmol/L to 2 nmol/L is also obtained.Moreover,by designing different stem-loop DNAs specific to different mi RNA targets and labeling them with different fluorophores,multiplexed mi RNAs can be simultaneously detected in one-tube reaction with the synchronous fluorescence spectrum(SFS)technique.

Acoustic emission detection using intensity-modulated DFB fiber laser sensor

The bonded distributed feedback（DFB） fiber laser（FL） acoustic emission sensor and the intensity response of the DFB-FL to external acoustic emissions are investigated. The dynamic sensitivity of the DFB-FL is calibrated by a referenced piezoelectric receiver. In the DFB-FL we used here, the minimum detectable signal is2 × 10^（-6）m∕s at 5 kHz. Using wavelet packet technology, the collected signals are analyzed, which confirms that an intensity-modulated DFB-FL sensor can be used to detect acoustic emission signals.

A photopatterned SERS substrate with a sandwich structure for multiplex detection

Substrate photopatterning has provided versatile applications in biomedical fields. Herein, an universal and efficient photoligation reaction has been used to prepare a patterned capture substrate for a sandwich SERS immunoassay. Photoirradiation induces mild and efficient immobilization of antibodies at the desired region of a gold surface, and the antibody-antigen interaction helps the substrate to capture the antigens in solution specifically. After exposing to SERS probes, i.e., the gold nanoparticles labelled with both antibodies and intrinsically strong Raman reporters, multiple quantitative SERS determination of antigens can be achieved with high sensitivity and specificity. The limit of detection can be as low as 10^(-12) mol/L for four kinds of cancer biomarkers, which provides a promising method for the construction of highly sensitive and high-throughput SERS detection chip and the application of in vitro diagnosis.

Transmission of 200 Tb/s （375 × 3 × 178.125 Gb/s） PDM-DFTS-OFDM-32QAM super channel over lkm FMF

A few-mode fiber （FMF） is designed to support three spatial modes （LP01, LP 11a, and LP 11 b） and fabricated through plasma chemical vapor deposition （PCVD）and rod-in-tube （RIT） method. Using PDM-DFTS-OFDM- 32QAM modulation, wavelength division multiplexing, mode multiplexing, and coherent detection, we successfully demonstrated 200Tb/s （375× 3 × 178.125Gb/s） signal over 1 km FMF using C and L bands with 25 GHz channel spacing. After 1 km FMF transmission, all the tested bit error rates （BERs） are below 20% forward error correction （FEC） threshold （2.0 × 10-2）. Within each sub-channel, we achieved a spectral efficiency of 21.375 bits/Hz in the C and L bands.