Bladder cancer risk stratification with the Oncuria 10-plex bead-based urinalysis assay using three different Luminex xMAP instrumentation platforms.

BACKGROUND: No single marker of bladder cancer (BC) exists in urine samples with sufficient accuracy for disease diagnosis and treatment monitoring. The multiplex Oncuria BC assay noninvasively quantifies the concentration of 10 protein analytes in voided urine samples to quickly generate a unique molecular profile with proven BC diagnostic and treatment-tracking utility. Test adoption by diagnostic and research laboratories mandates reliably reproducible assay performance across a variety of instrumentation platforms used in different laboratories. METHODS: We compared the performance of the clinically validated Oncuria BC multiplex immunoassay when data output was generated on three different analyzer systems. Voided urine samples from 36 subjects (18 with BC and 18 Controls) were reacted with Oncuria test reagents in three 96-well microtiter plates on Day 1, and consecutively evaluated on the LED/image-based MagPix, and laser/flow-based Luminex 200 and FlexMap 3D (all xMAP instruments from Luminex Corp., Austin, TX) on Day 2. The BC assay uses magnetic bead-based fluorescence technology (xMAP, Multi-analyte profiling; Luminex) to simultaneously quantify 10 protein analytes in urine specimens [i.e., angiogenin (ANG), apolipoprotein E (ApoE), carbonic anhydrase IX (CA9), CXCL8/interleukin-8 (IL-8), matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-10 (MMP-10), serpin A1/alpha-1 anti-trypsin (A1AT), serpin E1/plasminogen activator inhibitor-1 (PAI-1), CD138/syndecan-1 (SDC1), and vascular endothelial growth factor-A (VEGF-A)]. All three analyzers quantify fluorescence signals generated by the Oncuria assay. RESULTS: All three platforms categorized all 10 analytes in identical samples at nearly identical concentrations, with variance across systems typically < 5%. While the most contemporary instrument, the FlexMap 3D, output higher raw fluorescence values than the two comparator systems, standard curve slopes and analyte concentrations determined in urine samples were concordant across all three units. Forty-four percent of BC samples registered ≥ 1 analyte above the highest standard concentration, i.e., A1AT (n = 7/18), IL-8 (n = 5), and/or ANG (n = 2), while only one control sample registered an analyte (A1AT) above the highest standard concentration. CONCLUSION: Multiplex BC assays generate detailed molecular signatures useful for identifying BC, predicting treatment responsiveness, and tracking disease progression and recurrence. The similar performance of the Oncuria assay across three different analyzer systems supports test adaptation by clinical and research laboratories using existing xMAP platforms. TRIAL REGISTRATION: This study was registered at ClinicalTrials.gov as NCT04564781, NCT03193528, NCT03193541, and NCT03193515.

Rapid and sensitive lateral flow biosensor for the detection of GII human norovirus based on immunofluorescent nanomagnetic microspheres.

Human norovirus (HuNoV) is the most predominant viral agents of acute gastroenteritis. Point-of-care testing (POCT) based on lateral flow immunochromatography (LIFC) has become an important tool for rapid diagnosis of HuNoVs. However, low sensitivity and lack of quantitation are the bottlenecks of traditional LIFC. Thus, we established a rapid and accurate technique that combined immunomagnetic enrichment (IM) with LFIC to identify GII HuNoVs in fecal specimens. Before preparing immunofluorescent nanomagnetic microspheres and achieving the effect of HuNoV enrichment in IM and fluorescent signal in LFIC, amino-functionalized magnetic beads (MBs) and carboxylated quantum dots (QDs) were coupled at a mass ratio of 4:10. Anti-HuNoV monoclonal antibody was then conjugated with QDs-MB. The limit of detection was 1.56 × 104 copies/mL, and the quantitative detection range was 1.56 × 104 copies/mL-1 × 106 copies/mL under optimal circumstances. The common HuNoV genotypes GII.2, GII.3, GII.4, and GII.17 can be detected, there was no cross-reaction with various enteric viruses, including rotavirus, astrovirus, enterovirus, and sapovirus. A comparison between IM-LFIC and RT-qPCR for the detection of 87 fecal specimens showed a high level of agreement (kappa = 0.799). This suggested that the method is rapid and sensitive, making it a promising option for point-of-care testing in the future.

NT-proBNP detection with a one-step magnetic lateral flow channel assay.

Point-of-care sensors targeting blood marker analysis must be designed to function with very small volumes since acquiring a blood sample through a simple, mostly pain-free finger prick dramatically limits the sample size and comforts the patient. Therefore, we explored the potential of converting a conventional lateral flow assay (LFA) for a significant biomarker into a self-contained and compact polymer channel-based LFA to minimize the sample volume while maintaining the analytical merits. Our primary objective was to eliminate the use of sample-absorbing fleece and membrane materials commonly present in LFAs. Simultaneously, we concentrated on developing a ready-to-deploy one-step LFA format, characterized by dried reagents, facilitating automation and precise sample transport through a pump control system. We targeted the detection of the heart failure biomarker NT-proBNP in only 15 µL human whole blood and therefore implemented strategies that ensure highly sensitive detection. The biosensor combines streptavidin-functionalized magnetic beads (MNPs) as a 3D detection zone and fluorescence nanoparticles as signal labels in a sandwich-based immunoassay. Compared to the currently commercialized LFA, our biosensor demonstrates comparable analytical performance with only a tenth of the sample volume. With a detection limit of 43.1 pg∙mL-1 and a mean error of 18% (n ≥ 3), the biosensor offers high sensitivity and accuracy. The integration of all-dried long-term stable reagents further enhances the convenience and stability of the biosensor. This lateral flow channel platform represents a promising advancement in point-of-care diagnostics for heart failure biomarkers, offering a user-friendly and sensitive platform for rapid and reliable testing with low finger-prick blood sample volumes.

An Alternative Application of Magnetic-Activated Cell Sorting: CD45 and CD235a Based Purification of Semen and Testicular Tissue Samples.

Magnetic activated cell sorting (MACS) is a well-known sperm selection technique, which is able to remove apoptotic spermatozoa from semen samples using the classic annexinV based method. Leukocytes and erythrocytes in semen samples or in testicular tissue processed for in vitro fertilization (IVF) could exert detrimental effects on sperm. In the current study, we rethought the aforementioned technique and used magnetic microbeads conjugated with anti-CD45/CD235a antibodies to eliminate contaminating leukocytes and erythrocytes from leukocytospermic semen samples and testicular tissue samples gained via testicular sperm extraction (TESE). With this technique, a 15.7- and a 30.8-fold reduction could be achieved in the ratio of leukocytes in semen and in the number of erythrocytes in TESE samples, respectively. Our results show that MACS is a method worth to reconsider, with more potential alternative applications. Investigations to find molecules labeling high-quality sperm population and the development of positive selection procedures based on these might be a direction of future research.

Separation mixed semen of two individuals using magnetic beads coupled ABH blood group antibody.

In sexual assault cases, one of the most common samples collected is a mixed semen stain, which is often found on the vagina, female underwear, or bed sheets. However, it is usually difficult to identify the perpetrator based on this sample alone. One technique that has been developed to address this issue is magnetic bead-based separation. This method involves using modified magnetic microspheres to capture and enrich specific target cells, in this case, sperm cells. In this study, we utilized magnetic beads coupled with ABH blood group antibody to isolate sperm cells from an individual of a single ABO blood type. Subsequently, polymerase chain reaction amplification and capillary electrophoresis were employed to perform the genotyping the short tandem repeat (STR) loci. This approach allows for the identification of different individuals in a mixed seminal stain sample from two individuals, by first separating sperm cells based on ABH antigen differences and subsequently utilizing autosomal STR typing on the enriched single blood group cells.

Separation and colorimetric detection of Escherichia coli by phage tail fiber protein combined with nano-magnetic beads.

A colorimetric detection method for Escherichia coli (E. coli) in water was established based on a T7 phage tail fiber protein-magnetic separation. Firstly, the tail fiber protein (TFP) was expressed and purified to specifically recognize E. coli, which was verified by using fusion protein GFP-tagged TFP (GFP-TFP) and fluorescence microscopy. Then TFP conjugated with magnetic beads were applied to capture and separate E. coli. The TFP was covalently immobilized on the surface of magnetic beads and captured E. coli as verified by scanning electron microscopy (SEM). Finally, polymyxin B was used to lyse E. coli in solution and the released intracellular ß-galactosidase (ß-gal) could hydrolyze the colorimetric substrate chlorophenol red-ß-D-galactopyranoside (CPRG), causing color change from yellow to purple. The high capture efficiencies of E. coli ranged from 88.70% to 95.65% and E. coli could be detected at a concentration of 102 CFU/mL by naked eyes. The specificity of the chromogenic substrate was evaluated using five different pathogen strains as competitors and tests with four kinds of real water samples showed recoveries of 86.00% to 92.25%. The colorimetric changes determined by visual inspection can be developed as an efficient platform for point-of-care detection of E. coli in resource-limited regions.

Differentiated expressed miRNAs in splenic monocyte induced by burn injury in mice.

To find potential biomarkers based on miRNA and their potential targets in splenic monocytes in burn-injured mice. Male Balb/c mice were subjected to sham or scalding injury of 15% total body surface area. Spenic CD11b+ monocytes were purified with magnetic beads. The monocytes were cultured in the presence of lipopolysaccharide. The proliferation of monocytes was detected by MTT assay, and the cytokines in the supernatant were examined by enzyme linked immunosorbent assay. The purified monocytes were also under total RNA extraction. The differential monocytic miRNAs expression between the sham and burn-injured mice was analysed by miRNA microarray. The activity of monocytes was comparable between the two groups (p > 0.05). However, monocytes from burn-injured mice secreted higher levels of tumour necrosis factor (TNF)-α and transforming growth factor-ß, but lower level of monocyte chemoattratctant protein-1. A total of 54 miRNAs were differentially expressed in monocytes from burn relative to sham-injured mice (fold >3). Further quantitative reverse transcription polymerase chain reaction confirmed that the expression of miR-146a was significantly down-regulated, while miR-3091-6p was up-regulated after burn injury. Using the combination of Miranda and TargetScan softwares, we found that mir-146a may regulate 180 potential target genes including TNF receptor related factor 6 (TRAF6), interleukin-1 receptor related kinase 1 (IRAK1) and CD28. Mir-3091-6p may regulate 39 potential targets, including SOCS7 (cytokine signal transduction inhibitor 7) and ARRB2 (arrestin, ß 2). The miRNAs expressed by monocytes after burn injury may be involved in the regulation of innate immune response in burn injury.

An automated magnetic bead extraction method for measuring plasma metanephrines and 3-methoxytyramine using liquid chromatography tandem mass spectrometry.

Liquid chromatography tandem mass spectrometry (LC-MS/MS) is used routinely in clinical diagnostics; however, automating the sample pretreatment is challenging. We established and evaluated an automated method based on the magnetic bead extraction principle (MBE) to measure normetanephrine (NMN), metanephrine (MN), and 3-methoxytyramine (3-MT). The target analytes were extracted, purified, and concentrated using different solvents and chemical bond-modified magnetic beads transferred via a magnetic bar. The linearity, recovery, matrix effect, and precision of MBE were evaluated thoroughly, and compared with traditional solid-phase extraction (SPE) using 131 plasma samples. The chromatography peaks of metanephrines and 3-MT, extracted via MBE, are symmetrical, without interfering peaks. The linearity was excellent with correlation coefficient (r) > 0.99. The MBE exhibited good reproducibility with within-run coefficient variations (CVs) of 1.96-2.00%, 4.06-5.75%, and 3.89-4.90% for MN, NMN, and 3-MT, respectively. The total CVs for MN, NMN, and 3-MT were 1.96-2.80%, 5.12-5.75%, and 5.44-6.27%, respectively. The relative recoveries for MN, NMN, and 3-MT varied between 93.5 and 107.4%, whereas their biases were all within 10%. The results for MN, NMN, and 3-MT extracted via MBE compared with SPE exhibited excellent correlation, with r > 0.99; the mean bias% for MN, NMN, and 3-MT were small (-2.9%, -3.2%, and -3.2%, respectively). In conclusion, the automated MBE method for measuring plasma metanephrines and 3-MT can be applied in future routine clinical diagnostics, and the MBE principle may indicate a new era for LC-MS/MS in clinical application.

Specific and sensitive detection of Influenza A virus using a biotin-coated nanoparticle enhanced immunomagnetic assay.

This study reports the development of a sensitive magnetic bead-based enzyme-linked immunoassay (MELISA) for the pan-reactive detection of the Influenza A virus. The assay combines immunomagnetic beads and biotin-nanoparticle-based detection to quantify a highly conserved viral nucleoprotein in virus lysates. At the capture step, monoclonal antibody-coated magnetic microbeads were used to bind and concentrate the nucleoprotein in samples. The colorimetric detection signal was amplified using biotinylated silica nanoparticles (NP). These nanoparticles were functionalized on the surface with short DNA spacers bearing biotin groups by an automated supported synthesis method performed on nano-on-micro assemblies with a DNA/RNA synthesizer. A biotin-nanoparticle and immunomagnetic bead-based assay was developed. We succeeded in detecting Influenza A viruses directly in the lysis buffer supplemented with 10% saliva to simulate the clinical context. The biotin-nanoparticle amplification step enabled detection limits as low as 3 × 103 PFU mL-1 and 4 × 104 PFU mL-1 to be achieved for the H1N1 and H3N2 strains respectively. In contrast, a classical ELISA test based on the same antibody sandwich showed detection limit of 1.2 × 107 PFU mL-1 for H1N1. The new enhanced MELISA proved to be specific, as no cross-reactivity was found with a porcine respiratory virus (PRRSV). Graphical abstract.

Prospects of NIR fluorescent nanosensors for green detection of SARS-CoV-2.

The pandemic of the novel coronavirus disease 2019 (COVID-19) is continuously causing hazards for the world. Effective detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can relieve the impact, but various toxic chemicals are also released into the environment. Fluorescence sensors offer a facile analytical strategy. During fluorescence sensing, biological samples such as tissues and body fluids have autofluorescence, giving false-positive/negative results because of the interferences. Fluorescence near-infrared (NIR) nanosensors can be designed from low-toxic materials with insignificant background signals. Although this research is still in its infancy, further developments in this field have the potential for sustainable detection of SARS-CoV-2. Herein, we summarize the reported NIR fluorescent nanosensors with the potential to detect SARS-CoV-2. The green synthesis of NIR fluorescent nanomaterials, environmentally compatible sensing strategies, and possible methods to reduce the testing frequencies are discussed. Further optimization strategies for developing NIR fluorescent nanosensors to facilitate greener diagnostics of SARS-CoV-2 for pandemic control are proposed.

Sensitive and reproducible detection of SARS-CoV-2 using SERS-based microdroplet sensor.

Surface-enhanced Raman scattering (SERS)-based assays have been recently developed to overcome the low detection sensitivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SERS-based assays using magnetic beads in microtubes slightly improved the limit of detection (LoD) for SARS-CoV-2. However, the sensitivity and reproducibility of the method are still insufficient for reliable SARS-CoV-2 detection. In this study, we developed a SERS-based microdroplet sensor to dramatically improve the LoD and reproducibility of SARS-CoV-2 detection. Raman signals were measured for SERS nanotags in 140 droplets passing through a laser focal volume fixed at the center of the channel for 15 s. A comparison of the Raman signals of SERS nanotags measured in a microtube with those measured for multiple droplets in the microfluidic channel revealed that the LoD and coefficient of variation significantly improved from 36 to 0.22 PFU/mL and 21.2% to 1.79%, respectively. This improvement resulted from the ensemble average effects because the signals were measured for SERS nanotags in multiple droplets. Moreover, the total assay time decreased from 30 to 10 min. A clinical test was performed on patient samples to evaluate the clinical efficacy of the SERS-based microdroplet sensor. The assay results agreed well with those measured by the reverse transcription-polymerase chain reaction (RT-PCR) method. The proposed SERS-based microdroplet sensor is expected to be used as a new point-of-care diagnostic platform for quick and accurate detection of SARS-CoV-2 in the field.

Phenol biodegradation by immobilized Rhodococcus qingshengii isolated from coking effluent on Na-alginate and magnetic chitosan-alginate nanocomposite.

Phenol is a hazardous organic solvent to living organisms, even in its small amounts. In order to bioremediation of phenol from aqueous solution, a novel bacterial strain was isolated from coking wastewater, identified as Rhodococcus qingshengii based on 16S rRNA sequence analysis and named as strain Sahand110. The phenol-biodegrading capabilities of the free and immobilized cells of Sahand110 on the beads of Na-alginate (NA) and magnetic chitosan-alginate (MCA) nanocomposite were evaluated under different initial phenol concentrations (200, 400, 600, 800 and 1000 mg/L). Results illustrated that Sahand110 was able to grow and complete degrade phenol up to 600 mg/L, as the sole carbon and energy source. Immobilized cells of Sahand110 on NA and MCA were more competent than its free cells in degradation of high phenol concentrations, 100% of 1000 mg/L phenol within 96 h, indicating the improved tolerance and performance of the immobilized cells against phenol toxicity. Therefore, the immobilized Sahand110 on the studied beads, especially MCA bead regarding its suitable properties, has significant potential to enhanced bioremediation of phenol-rich wastewaters.

Effect of a subset of adipose-derived stem cells isolated with liposome magnetic beads to promote cartilage repair.

This study aimed to investigate the ability of CD146+ subset of ADSCs to repair cartilage defects. In this study, we prepared CD146+ liposome magnetic beads (CD146+ LMB) to isolate CD146+ ADSCs. The cells were induced for chondrogenic differentiation and verified by cartilage-specific mRNA and protein expression. Then a mouse model of cartilage defect was constructed and treated by filling the induced cartilage cells into the damaged joint, to evaluate the function of such cells in the cartilage microenvironment. Our results demonstrated that the CD146+ LMBs we prepared were uniform, small and highly stable, and cell experiments showed that the CD146+ LMB has low cytotoxicity to the ADSCs. ADSCs isolated with CD146+ LMB were all CD146+ , CD105+ , CD166+ and CD73+ . After chondrogenic induction, the cells showed significantly increased expression of cartilage markers Sox9, collagen Ⅱ and aggrecan at protein level and significantly increased Sox9, collagen Ⅱ and aggrecan at mRNA level, and the protein expression and mRNA expression of CD146+ ADSCs group were higher than those of ADSCs group. The CD146+ ADSCs group showed superior tissue repair ability than the ADSCs group and blank control group in the animal experiment, as judged by gross observation, histological observation and histological scoring. The above results proved that CD146+ LMB can successfully isolate the CD146+ ADSCs, and after chondrogenic induction, these cells successfully promoted repair of articular cartilage defects, which may be a new direction of tissue engineering.

Magnetoresistance Properties of Red Blood Cells in Plasma Combined with Several Magnetic Beads Passing Two Cu Electrodes.

In recent years, research has been intensively carried out on the applicability of magnetic beads (MBs) and magnetic nanoparticles coupled to biological objects such as red blood cells (RBCs). The magnetoresistance (MR) of a solution of RBCs and MBs (RBCs+MBs) was evaluated when MBs migrated in the presence or absence of an external magnetic field. The pattern of distribution of the MBs, which were homogeneously suspended in deionized distilled water, varied depending on the magnitude of the external magnetic field applied between the Cu electrodes connected to the two terminals. As the magnitude of the external magnetic field is increased or decreased, MBs are split on both sides and evenly mixed, respectively. The ratios (ΔMR/MR) versus an external magnetic field for the solutions of only MBs and a mixed RBCs+MBs were -33.4% and -27.4% at ±30 Oe and ±46 Oe of coercive fields, respectively. These results show that a solution of RBCs+MBs can act like a high-resolution biosensor that detects the oxygenation state of RBCs.

Vancomycin-modified poly-l-lysine magnetic separation combined with multiplex polymerase chain reaction assay for efficient detection of Bacillus cereus in milk.

In this study, a new vancomycin (Van)-modified poly-l-lysine (PLL) magnetic bead (MB) technique was developed for isolation of gram-positive bacteria. The method combines magnetic separation with a multiplex PCR (mPCR) assay and gel electrophoresis for easy and rapid detection of Bacillus cereus. Vancomycin was used as a molecular ligand between the MB and the d-alanyl-d-alanine moieties on the cell wall surface of B. cereus. The PLL served as a flexible molecular tether between the MB and Van that reduced steric hindrance maintaining the biological activity of Van. The MB-PLL-Van capture nanoprobes exhibited excellent capture and isolation efficiency for B. cereus in spiked milk matrix samples without interference from the complex food matrix. The subsequent mPCR assay showed high specificity for the 4 target genes in B. cereus, the entFM, cesB, cer, and 16S rRNA genes, that were used to achieve efficient genotyping and detection. Under optimum conditions, the limit of detection reached 103 cfu/mL, with a dynamic range of detection at 103 to 107 cfu/mL in pure culture. Application of the MB-PLL-Van mediated mPCR assay for B. cereus in milk matrix samples achieved results similar to those of the pure culture. In addition, with a 6-h pre-enrichment of B. cereus that was spiked in milk matrix samples, the limit of detection reached 101 cfu/mL. The MB-PLL-Van mediated mPCR assay developed in this study could be used as a universal technology platform for the efficient enrichment and genotyping of gram-positive bacteria.

Poly-l-lysine-functionalized magnetic beads combined with polymerase chain reaction for the detection of Staphylococcus aureus and Escherichia coli O157:H7 in milk.

Rapid and credible detection of pathogens is essential to prevent and control outbreaks of foodborne diseases. In this study, a poly-l-lysine-functionalized magnetic beads (PLL-MB) strategy combined with a PCR assay was established to detect Staphylococcus aureus. We also detected Escherichia coli O157:H7 to further verify the strategy for gram-negative bacteria detection. Poly-l-lysine has strong positive charges because of its amino groups, which can conjugate with the carboxyl of carboxyl magnetic beads. Furthermore, it can be used to combine with bacteria through electrostatic adsorption. Under optimum conditions, the developed PLL-MB complexes showed 90% capture efficiency in phosphate-buffered saline and 85% capture efficiency in milk for S. aureus detection. The limit of detection of the PLL-MB-PCR assay was 102 cfu/mL (1.8 × 102 cfu/mL for S. aureus and 7 × 102 cfu/mL for E. coli O157:H7) in phosphate-buffered saline and milk samples. The whole assay can be performed within 4 h. The proposed strategy showed a lower limit of detection when compared with the conventional PCR assay without enrichment. In addition, this method exhibited the advantages of a high-efficient, cost-efficient, and simple operation, indicating its potential applications in foodborne pathogen detection.

Detection of biotin with zeptomole sensitivity using recombinant spores and a competition assay.

Detection of protein-binding analytes is important for many applications. Currently, various instrument-based techniques are used for detecting protein-binding analytes. However, such techniques have several limitations including high cost and time-consuming sample processing. In order to overcome these limitations, we developed a sensitive competition assay for the detection of protein-binding analytes using recombinant endospores as a sensing element. The method is based on the competition between the biotin, the model analyte, and a biotin-magnetic bead complex to bind the recombinant spores containing the biotin binding region of streptavidin. After magnetic attraction, the residual spores in the suspension are spread on plates to form colonies which are used to count the amount of the residual spores; the higher the residual ratio of spores, the more biotin in the samples. The linear range was from 150 zmol to 1.5 fmol and the limit of detection of the assay was 150 zmol. The assay proposed herein is sensitive and does not require any expensive equipment. It is suitable for qualitative or semi-quantitative analysis such as screening tests for the detection of toxic chemicals.

Deterministic Lateral Displacement-Based Separation of Magnetic Beads and Its Applications of Antibody Recognition.

This work presents a magnetic-driven deterministic lateral displacement (m-DLD) microfluidic device. A permanent magnet located at the outlet of the microchannel was used to generate the driving force. Two stages of mirrored round micropillar array were designed for the separation of magnetic beads with three different sizes in turn. The effects of the forcing angle and the inlet width of the micropillar array on the separating efficiency were studied. The m-DLD device with optimal structure parameters shows that the separating efficiencies for the 10 µm, 20 µm and 40 µm magnetic beads are 87%, 89% and 94%, respectively. Furthermore, this m-DLD device was used for antibody recognition and separation among a mixture solution of antibodies. The trajectories of different kinds of magnetic beads coupled with different antigens showed that the m-DLD device could realize a simple and low-cost diagnostic test.

Microfluidic-Based Detection of AML-Specific Biomarkers Using the Example of Promyelocyte Leukemia.

A microfluidic assay for the detection of promyelocytic leukemia (PML)-retinoic acid receptor α (RARα) fusion protein was developed. This microfluidic-based system can be used for rapid personalized differential diagnosis of acute promyelocyte leukemia (APL) with the aim of early initiation of individualized therapy. The fusion protein PML-RARα occurs in 95% of acute promyelocytic leukemia cases and is considered as diagnostically relevant. The fusion protein is formed as a result of translocation t(15,17) and is detected in the laboratory by fluorescence in situ hybridization (FISH) or reverse transcriptase polymerase chain reaction (RT-PCR). Diagnostic methods require many laboratory steps with specialized staff. The developed microfluidic assay includes a sandwich enzyme-linked immunosorbent assay (ELISA) system for PML-RARα on surface of magnetic microparticles in a microfluidic chip. A rapid detection of PML-RARα in cell lysates is achieved in less than one hour. A biotinylated PML-antibody on the surface of magnetic streptavidin coated microparticles is used as capture antibody. The bound translocation product is detected by a RARα antibody conjugated with horseradish peroxidase and the substrate QuantaRed. The analysis is performed in microfluidic channels which involves automated liquid processing with stringent washing and short incubation times. The results of the developed assay show that cell lysates of PML-RARα-positive cells (NB-4) can be clearly distinguished from PML-RARα-negative cells (HL-60, MV4-11).

Detection of potential biomarkers associated with outrageous diseases and environmental pollutants by nanoparticle-based immuno-PCR assays.

Immuno-polymerase chain reaction (I-PCR) assay with advantages of both enzyme-linked immunosorbent assay (ELISA) and PCR exhibits several-fold enhanced sensitivity in comparison to respective ELISA, which has wide applications for ultralow detection of several molecules, i.e. cytokines, protooncogenes and biomarkers associated with several diseases. Conjugation of reporter DNA to the detection antibodies is the most crucial step of I-PCR assay that usually employs streptavidin-protein A, streptavidin-biotin conjugate or succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) system by a covalent binding. However, coupling of antibodies and oligonucleotides to nanoparticles (NPs) is relatively easier in the NP-based I-PCR (NP-I-PCR) that also displays better accuracy. This article is mainly focused on the detection of important biomarkers associated with several outrageous infectious and non-infectious diseases by NP-I-PCR assays, which would expedite an early initiation of therapy thus human health would be improved. Similarly, ultralow detection of environmental pollutants by these assays and their elimination would certainly improve human health.