Total Assay Time Research Articles

Proposal of a Rapid Detection System Using Image Analysis for ELISA with an Autonomous Centrifugal Microfluidic System.

In this study, with the aim of adapting an enzyme-linked immunosorbent assay (ELISA) system for point-of-care testing (POCT), we propose an image analysis method for ELISAs using a centrifugal microfluidic device that automatically executes the assay. The developed image analysis method can be used to quantify the color development reaction on a TMB (3,3',5,5'-tetramethylbenzidine) substrate. In a conventional ELISA, reaction stopping reagents are required at the end of the TMB reaction. In contrast, the developed image analysis method can analyze color in the color-developing reaction without a reaction stopping reagent. This contributes to a reduction in total assay time. The microfluidic devices used in this study could execute reagent control for ELISAs by steady rotation. In the demonstration of the assay and image analysis, a calibration curve for mouse IgG detection was successfully prepared, and it was confirmed that the image analysis method had the same performance as the conventional analysis method. Moreover, the changes in the amount of color over time confirmed that a calibration curve equal to the endpoint analysis was obtained within 2 min from the start of the TMB reaction. As the assay time before the TMB reaction was approximately 7.5 min, the developed ELISA system could detect TMB in just 10 min. In conventional methods using a plate reader, the assay required a time of 90 min for manual handling using microwell plates, and in the case of using automatic microfluidic devices, 30 min were required. The time of 10 min realized by this proposed method is equal to the time required for detection in an immunochromatographic assay with a lateral flow assay; therefore, it is expected that ELISAs can be performed sufficiently to adapt to POCT.

NanoString nCounter-Based Assay for Detection of Fusion-Associated Salivary Gland Tumors.

Salivary gland tumors include numerous subtypes that vary from benign to highly aggressive, with many showing overlapping histopathological features that can make diagnosis challenging. Most subtypes express driver fusion genes that are tumor specific, and detection of such fusions is useful for differentiating amongst specific diagnoses, determining appropriate tumor grading, and guiding effective treatment. Currently, fusions can be detected by FISH, RT-PCR or through next-generation sequencing approaches, all of which are highly effective methodologies but can be costly or time consuming. We developed a rapid NanoString nCounter platform-based assay to detect salivary gland tumor fusions using a combination of fusion junction-specific probes and an approach through differential exon expression analysis. The assay includes 68 junction-specific probes and analysis of exon expression across 9 fusion-associated genes in a single multiplex assay. Out of 55 retrospective and 171 prospective cases assayed, we accurately detected the majority of cases of pleomorphic adenoma, adenoid cystic carcinoma, cribriform adenocarcinoma, clear cell carcinoma, secretory carcinoma and NUT-rearranged carcinoma, including cases of these tumor types arising in non-salivary gland sites, with the major drawback being an inability to detect MAML2-containing mucoepidermoid samples. With mucoepidermoid carcinoma excluded, the assay shows an overall sensitivity of 96.1% and specificity of 100%. We show that the majority of salivary gland tumor fusions can be effectively detected with a single rapid NanoString based assay, which can serve as a useful adjunctive tool for routine diagnostic head and neck pathology. The assay is low cost with a rapid turnaround time (30h total assay time per sample batch, with minimal technician input required) compared to alternate detection methods.

Anti-OTC antibody-conjugated fluorescent magnetic/silica and fluorescent hybrid silica nanoparticles for oxytetracycline detection

Abstract This study presents two alternative fluorescent nanoparticle-based oxytetracycline (OTC) detection methods in milk samples. Rhodamine 6G-coated fluorescent hybrid silica nanoparticles and fluorescent magnetic/silica nanoparticles functionalized with anti-OTC antibodies were used in this test. The sandwich test format was utilized to compare anti-OTC antibody-conjugated fluorescent magnetic/silica nanoparticles with OTC/OTC antibody-conjugated fluorescent hybrid silica nanoparticles in an Eppendorf tube with magnetic separators. The magnetic separator helps to quickly retain all of the OTC captured by fluorescent magnetic core–shell nanoparticles in the milk sample. As a result, the assay time was dramatically shortened. The obtained linear range was 1.34 × 10−6 to 2.10 × 10−8 (M) (R 2 = 0.9954), the detection limit was 4.76 ng/mL, and the total assay time was 90 min. This approach was used to determine the OTC concentration in milk samples, and the maximum percentage (%) of interference was less than 3.0%, with a recovery rate of greater than 97.0%. This approach offers a high potential for residue detection in milk samples. With a total analysis period of less than 90 min, this approach provided the best way to determine the capture and detector nanoparticles’ response.

A novel integrated lateral flow immunoassay platform for the detection of cardiac troponin I using hierarchical dendritic copper-nickel nanostructures

Cardiac troponin I (cTnI) is a critical biomarker for the diagnosis of acute myocardial infarction (AMI). Herein, we report a novel integrated lateral flow immunoassay (LFIA) platform for highly sensitive point-of-care testing (POCT) of cTnI using hierarchical dendritic copper-nickel (HD-nanoCu-Ni) nanostructures. The electrodeposited HD-nanoCu-Ni film (∼22 μm thick) on an ITO-coated glass substrate exhibits superior capillary action and structural integrity. These properties enable efficient sample transport and antibody immobilization, making it a compelling alternative to conventional multi-component paper-based LFIA test strips, which are often plagued by structural fragility and susceptibility to moisture damage. The biofunctionalized HD-nanoCu-Ni substrates were laser-etched with lateral flow channels, including a sample loading/conjugate release zone, a test zone, and a control zone. Numerical simulations were used to further optimize the design of these channels to achieve optimal fluid flow and target capture. The HD-nanoCu-Ni LFIA device utilizes a fluorescence quenching based sandwich immunoassay format using antibody-labeled gold nanoparticles (AuNPs) as quenchers. Two different fluorescent materials, fluorescein isothiocyanate (FITC) and CdSe@ZnS quantum dots (QDs), were used as background fluorophores in the device. Upon the formation of a sandwich immunocomplex with cTnI on the HD-nanoCu-Ni device, introduced AuNPs led to the fluorescence quenching of the background fluorophores. The total assay time was approximately 15 min, demonstrating the rapid and efficient nature of the HD-nanoCu-Ni LFIA platform. For FITC, both inner filter effect (IFE) and fluorescence resonance energy transfer (FRET) contributed to the AuNP-mediated quenching. In the case of CdSe@ZnS QDs, IFE dominated the AuNP-induced quenching. Calibration curves were established based on the relationship between the fluorescence quenching intensity and cTnI concentration in human serum samples, ranging from 0.5 to 128 ng/mL. The limits of detection (LODs) were determined to be 0.27 ng/mL and 0.40 ng/mL for FITC and CdSe@ZnS QDs, respectively. A method comparison study using Passing-Bablok regression analysis on varying cTnI concentrations in human serum samples confirmed the equivalence of the HD-nanoCu-Ni LFIA platform to a commercial fluorescence cTnI LFIA assay kit, with no significant systematic or proportional bias observed.

Membrane-Free Lateral Flow Assay with the Active Control of Fluid Transport for Ultrasensitive Cardiac Biomarker Detection.

Membrane-based lateral flow immunoassays (LFAs) have been employed as early point-of-care (POC) testing tools in clinical settings. However, the varying membrane properties, uncontrollable sample transport in LFAs, visual readout, and required large sample volumes have been major limiting factors in realizing needed sensitivity and desirable precise quantification. Addressing these challenges, we designed a membrane-free system in which the desirable three-dimensional (3D) structure of the detection zone is imitated and used a small pump for fluid flow and fluorescence as readout, all the while maintaining a one-step assay protocol. A hydrogel-like protein-polyelectrolyte complex (PPC) within a polyelectrolyte multilayer (PEM) was developed as the test line by complexing polystreptavidin (pSA) with poly(diallyldimethylammonium chloride) (PDDA), which in turn was layered with poly(acrylic acid) (PAA) resulting in a superior 3D streptavidin-rich test line. Since the remainder of the microchannel remains material-free, good flow control is achieved, and with the total volume of 20 μL, 7.5-fold smaller sample volumes can be used in comparison to conventional LFAs. High sensitivity with desirable reproducibility and a 20 min total assay time were achieved for the detection of NT-proBNP in plasma with a dynamic range of 60-9000 pg·mL-1 and a limit of detection of 56 pg·mL-1 using probe antibody-modified fluorescence nanoparticles. While instrument-free visual detection is no longer possible, the developed lateral flow channel platform has the potential to dramatically expand the LFA applicability, as it overcomes the limitations of membrane-based immunoassays, ultimately improving the accuracy and reducing the sample volume so that finger-prick analyses can easily be done in a one-step assay for analytes present at very low concentrations.

Detection of P. malariae using a new rapid isothermal amplification lateral flow assay

BackgroundWhile Plasmodium falciparum and Plasmodium vivax cause the majority of malaria cases and deaths, infection by Plasmodium malariae and other Plasmodium species also causes morbidity and mortality. Current understanding of these infections is limited in part by existing point-of-care diagnostics that fail to differentiate them and have poor sensitivity for low-density infections. Accurate diagnosis currently requires molecular assays performed in well-resourced laboratories. This report describes the development of a P. malariae diagnostic assay that uses rapid, isothermal recombinase polymerase amplification (RPA) and lateral-flow-strip detection.MethodsMultiple combinations of custom RPA primers and probes were designed using publicly available P. malariae genomic sequences, and by modifying published primer sets. Based on manufacturer RPA reaction conditions (TwistDx nfo kit), an isothermal assay was optimized targeting the multicopy P. malariae 18S rRNA gene with 39 °C incubation and 30-min run time. RPA product was visualized using lateral strips (FAM-labeled, biotinylated amplicon detected by a sandwich immunoassay, visualized using gold nanoparticles). Analytical sensitivity was evaluated using 18S rRNA plasmid DNA, and clinical sensitivity determined using qPCR-confirmed samples collected from Tanzania, Ethiopia, and the Democratic Republic of the Congo.ResultsUsing 18S rRNA plasmid DNA, the assay demonstrates a detection limit of 10 copies/µL (~ 1.7 genome equivalents) and 100% analytical specificity. Testing in field samples showed 95% clinical sensitivity and 88% specificity compared to qPCR. Total assay time was less than 40 min.ConclusionCombined with simplified DNA extraction methods, the assay has potential for future field-deployable, point-of-care use to detect P. malariae infection, which remains largely undiagnosed but a neglected cause of chronic malaria. The assay provides a rapid, simple readout on a lateral flow strip without the need for expensive laboratory equipment.

Performance of the digital cell morphology analyzer MC-100i in a multicenter study in tertiary hospitals in China

BackgroundThis study investigated the performance of the MC-100i, a pre-commercial digital morphology analyzer utilizing a convolutional neural network algorithm, in a multicentric setting involving up to 11 tertiary hospitals in China. MethodsBlood smears were analyzed by MC-100i, verified by morphologists, and manually differentiated. The classification performance on WBCs and RBCs was evaluated by comparing the classification results using different methods. The PLT and PLT clump counting performance was also assessed. The total assay time including hands-on time was evaluated. ResultsThe agreements between pre- and post-classification were high for normal WBCs (κ > 0.96) and lower for overall abnormal WBCs (κ = 0.90). The post-classification results correlated well with manual differentials for both normal and abnormal WBCs (r > 0.93), except for basophils (r = 0.8480) and atypical lymphocytes (r = 0.8211). The clinical sensitivity and specificity of each RBC abnormality after verification were above 90 % using microscopy reviews as the reference. The PLTs counted by the MC-100i before and after verification correlated well with those measured by the PLT-O mode (r = 0.98). Moreover, PLT clumps were successfully classified by the analyzer in EDTA-dependent pseudothrombocytopenia blood samples. ConclusionsThe MC-100i is an accurate and reliable digital cell morphology analyzer, offering another intelligent option for hematology laboratories.

A simplified lateral flow immunosensor for the assay of carcinoembryonic antigen in low-resource settings.

Carcinoembryonic antigen (CEA) is a glycoprotein widely used as a tumor marker. In this work, a colorimetric lateral flow immunosensor is developed for rapid and low-cost quantification of CEA in human blood serum. The immunosensor consists of a glass fiber sample/conjugation pad, a nitrocellulose detection pad and a cellulose absorption pad. The detection is based on a sandwich immunoreaction: the sample/conjugation pad is modified with gold nanoparticles (GNPs)-labeled anti-CEA conjugate probes which bind to the CEA target molecules in the sample and the complexes are captured at capture anti-CEA immobilized at the test line. The color intensity of the test line, measured from a scanned image of the strip, is related to the CEA concentration in the sample. The different assay parameters are studied in detail. The linearity holds from 1.25 to 640 ng mL-1 of CEA, the instrumental and visual limits of detection are 0.45 and 0.63 ng mL-1, respectively, and the total assay time is 15 min. The specificity of the immunoassay versus other cancer biomarkers is satisfactory. The recovery in samples of human serum spiked with CEA is in the range of 81-118% and the coefficient of variation of the method is ≤10%. Results obtained with the lateral flow immunosensor correlated well with a reference radioimmunoassay method (R2 = 0.99). This immunosensor can be readily applied to CEA monitoring at the point-of-care (POC) or in resource-limited settings thanks to its low-cost and simplicity.

SERS-based immunoassay on a plasmonic syringe filter for improved sampling and labeling efficiency of biomarkers.

Rapid, sensitive, and quantitative detection of biomarkers is needed for early diagnosis of disease and surveillance of infectious outbreaks. Here, we exploit a plasmonic syringe filter and surface-enhanced Raman spectroscopy (SERS) in the development of a rapid detection system, using human IgG as a model diagnostic biomarker. The novel assay design facilitates multiple passages of the sample and labeling solution through the detection zone enabling us to investigate and maximize sampling efficiency to the capture substrate. The vertical flow immunoassay process in this study involves the utilization of filter paper embedded with gold nanoparticles (AuNPs) to form a plasmonic substrate. Capture antibody (anti-human IgG) is then immobilized onto the prepared plasmonic paper and inserted into a vertical flow device (syringe filter holder). Sample solution is passed through the filter paper and the target antigen (human IgG) is selectively captured by the immobilized antibody to form an antibody-antigen complex. Next, functionalized AuNPs as extrinsic Raman labels (ERLs) are passed through the filter paper to label the captured biomarker molecules forming a layered structure. This sandwiched geometry enhances plasmonic coupling and SERS signal to provide highly sensitive detection of biomolecules. Systematic studies to investigate the impact of multiple infuse/withdraw cycles of the sample and labeling solutions reveal that antigen and ERL binding are maximized with 10 and 20 cycles, respectively. The optimized assay achieves a detection limit of ∼0.2 ng mL-1 for human IgG with a total assay time of less than 5 minutes, meeting the demands for rapid point of care diagnostics. Additionally, the optimized platform was implemented in the quantitative analysis of the SARS-CoV-2 nucleocapsid protein, the typical target in commercial, FDA-approved rapid antigen tests for COVID-19.

On-Particle Hyperbranched Rolling Circle Amplification-Scaffolded Magnetic Nanoactuator Assembly for Ferromagnetic Resonance Detection of MicroRNA.

Inspired by natural molecular machines, scientists are devoted to designing nanomachines that can navigate in aqueous solutions, sense their microenvironment, actuate, and respond. Among different strategies, magnetically driven nanoactuators can easily be operated remotely in liquids and thus are valuable in biosensing. Here we report a magnetic nanoactuator swarm with rotating-magnetic-field-controlled conformational changes for reaction acceleration and target quantification. By grafting nucleic acid amplification primers, magnetic nanoparticle (MNP) actuators can assemble and be fixed with a flexible DNA scaffold generated by surface-localized hyperbranched rolling circle amplification in response to the presence of a target microRNA, osa-miR156. Net magnetic anisotropy changes of the system induced by the MNP assembly can be measured by ferromagnetic resonance spectroscopy as shifts in the resonance field. With a total assay time of ca. 120 min, the proposed biosensor offers a limit of detection of 6 fM with a dynamic detection range spanning 5 orders of magnitude. The specificity of the system is validated by testing different microRNAs and salmon sperm DNA. Endogenous microRNAs extracted from Oryza sativa leaves are tested with both quantitative reverse transcription-PCR and our approach, showing comparable performances with a Pearson correlation coefficient >0.9 (n = 20).

Ag nanorod@PEI-Ag nanohybrid as an excellent signal label for sensitive and rapid detection of serum HER2

The accurate detection of Human epidermal growth factor receptor-2 (HER2) as a critical breast cancer biomarker can be essential for the early selection of therapeutic approaches. HER2 is a prominent component of a signaling network. Overexpression of the HER2 protein due to amplification of its gene leads to the development of an aggressive subtype of breast cancer. Patients with tumors that overexpress HER2 are eligible for treatment that significantly reduces mortality rates. Herein, we present a fast and simple method for detecting serum HER2. A new electrochemical label has been developed using charged Ag nanorod@ polyethylenimine-Ag (Ag NR@ PEI-Ag) nanohybrid. The synthesized Ag NR@PEI-Ag nanohybrid simultaneously has the electroactive property of silver and the large surface area of the PEI, which results in the enhancement of the detection signal. So, using Ag NR@PEI-Ag nanohybrid as the electrochemical label, a simple, fast, and sensitive electrochemical biosensor was designed to detect HER2. This way, after immobilizing HER2 aptamer on the Au electrode surface, HER2 or human serum was exposed to the aptamer. Then, the positively charged Ag NR@PEI-Ag nanohybrid was adsorbed onto the negatively charged aptamer-HER2 complex, and the current that was produced due to the Ag/AgCl reaction was measured as the electrochemical signal. The aptasensor shows a broad linear response from 10–12 to 10–7 g, a low detection limit (LOD) of 10 pg, and a total assay time of ~ 30 min.

Multienzymatic disintegration of DNA-scaffolded magnetic nanoparticle assembly for malarial mitochondrial DNA detection

Early diagnosis of malaria can prevent the spread of disease and save lives, which, however, remains challenging in remote and less developed regions. Here we report a portable and low-cost optomagnetic biosensor for rapid amplification and detection of malarial mitochondrial DNA. Bioresponsive magnetic nanoparticle assemblies are constructed by using nucleic acid scaffolds containing endonucleolytic DNAzymes and their substrates, which can be activated by the presence of target DNA and self-disintegrated to release magnetic nanoparticles for optomagnetic quantification. Specifically, target molecules can induce padlock probe ligation and subsequent one-pot homogeneous cascade reactions consisting of nicking-enhanced rolling circle amplification, DNAzyme-assisted nucleic acid recycling, and strand-displacement-driven disintegration of the magnetic assembly. With an optimized magnetic actuation process for reaction acceleration, a detection limit of 1 fM can be achieved by the proposed biosensor with a total assay time of ca. 90 min and a dynamic detection range spanning 3 orders of magnitude. The robustness of the system was validated by testing target molecules spiked in 5% serum samples. Clinical sample validation was conducted by testing malaria-positive clinical blood specimens, obtaining quantitative results concordant with qPCR measurements.

Electrochemical Biosensor for the Rapid Detection of HIV P24 Protein

Human immunodeficiency virus (HIV) is a deadly virus that propagates rapidly. It leads to acquired immunodeficiency syndrome (AIDS) disease with time. Early detection is one way to decrease the spread of the virus. P24 protein is the capsid protein of HIV available for detection in one’s body two weeks after the virus infection. A nickel-based electrochemical sensor is developed to detect the P24 protein using electrocatalysis. A rotating disk electrode is used to minimize the mass limitations. Also, the sensor is label-free which limits the dependence on immobilization. Cyclic voltammetry (CV), chronoamperometry, and electrochemical impedance spectroscopy (EIS) are used throughout the detection process. The electrochemical method CV is used to produce the catalyst layer on the nickel surface, which paves the way for the other two methods to monitor the current response and resistance of the P24 protein with the nickel surface. The sensor detects the presence of HIV P24 protein in the 1 ms of chronoamperometry. Also, the total assay time is less than other conventional methods. The sensor showed good sensitivity and a lower detection limit of around 70 ng/mL, which makes the sensor more reliable. Also, the sensor showed a linear current response with the increasing concentration of P24 protein. So, this simple biosensor has applications to decrease the infections of HIV.

A 3D-Printed Electrochemical Immunosensor Employing Cd/Se ZnS QDs as Labels for the Rapid and Ultrasensitive Detection of Salmonella typhimurium in Poultry Samples

Salmonella is one of the leading causes of foodborne illnesses worldwide, with poultry products being a major source of contamination. Thus, the detection of salmonella in commercial poultry products is crucial to minimize the effects on public health. Electrochemical sensors are promising tools for bacteria detection due to their sensitivity, simplicity, and potential for on-site analysis. In this work, a three-dimensional (3D) printed electrochemical immunosensor for the determination of Salmonella typhimurium in fresh chicken through a sandwich immunoassay employing biotinylated anti-S. typhimurium antibody followed by streptavidin labeled with Cd/Se ZnS quantum dots (QDs) is presented. The device features three carbon-black polylactic acid electrodes and a holder, and the quantification of S. typhimurium is performed by anodic stripping voltametric (ASV) determination of the Cd(II) released after acidic dissolution of the QDs. To enhance sensitivity, an electroplated bismuth film was deposited on the working electrode, achieving a detection limit of 5 cfu/mL in a total assay time of 25 min, whereas 5 h of sample pre-enrichment was required for the detection of 1 cfu/25 mL of chicken rinse and chicken broth. The method is accurate, with %recovery values ranging from 93.3 to 113% in fresh chicken samples, and repeatable with intra- and inter- assay coefficient of variations <2 and 5%, respectively, indicating the suitability of the proposed immunosensor for the detection of S. typhimurium at the point-of-need.

CRISPR-Cas12a-based aptasensor for sensitive and selective FB1 detection

Fumonisin B1 (FB1) is a common mycotoxin found in agricultural products. Therefore, establishing rapid detection methods is an important means to prevent the harm of FB1. This study was based on the clustered, regularly spaced short palindromic repeats (CRISPR)-Cas12a system with the introduction of aptamer F10. This study aimed to design CRISPR RNA (crRNA) to specifically recognize the aptamer F10, which formed the competitive relationship between the target molecule and crRNA for nucleic acid aptamer. Finally, a new type of biosensor was established using the trans-cleavage activity of Cas12a protein and amplifying the signal with a fluorescent probe. This study observed a linear relationship of FB1 concentrations in the range of 25–500 nM. The standard curve was y = −0.2586 × x + 775.3, R2 = 0.9922, and the limit of detection (LOD) was 16.84 nM. The recovery of the maize sample was 92.2–101.93% and 92.24–103.96%. The total assay time was 40 min. The biosensor showed high specificity and sensitivity for FB1 after the introduction of the aptamer F10, and the workflow was simple, convenient, and fast, which was suitable for point-of-care (POC) detection in the field. Our designed biosensor validated the principle of detecting small molecules using CRISPR-Cas12a systems, further facilitating its application in the diagnostic field.

A particle-based microfluidic fluorescent lateral flow assay for rapid and sensitive detection of SARS-CoV-2 antibody

Prompt isolation and treatment of patients are effective measures for preventing the spread of infectious diseases. Therefore, a sensitive and early diagnostic test is crucial for controlling pandemics. Herein, we present a tubeless integrated microfluidic lateral flow immunoassay chip for rapid and sensitive detection of SARS-CoV-2 antibodies. Fully automated chip operation is demonstrated by incorporating a dry reagent storage chamber, staggered herringbone micromixer, mobile particle-based detection zone, and spontaneous liquid transportation mechanism. The effects of the position and number of dry reagent patterns on assay time and detection sensitivity were investigated. Under laminar flow conditions, a staggered herringbone mixer was used to achieve rapid and complete mixing of the sample and reagent. In addition, we found that the volumetric flux of the sample and reagent complex over the surface of the particles is a crucial factor for improving detection sensitivity. The limit of detection of an anti-SARS-CoV-2 spike protein monoclonal antibody was determined to be 10.35 ng/mL in assay buffer with a total assay time of 15 min. In the analyte concentration range of 15.625–1000 ng/mL, the linearity and spike-recovery of the assay on serially diluted serum samples were excellent (R2 ≈ 0.99 and recovery rate of 82.9–112 %). The integrated microfluidic immunoassay chip demonstrates rapid and simple operation, which is promising for the diagnosis and monitoring of SARS-CoV-2 antibodies.

Synthesis and characterization of Au-decorated graphene oxide nanocomposite for magneto-electrochemical detection of SARS-CoV-2 nucleocapsid gene

Fast and effective diagnosis is the first step in monitoring the current coronavirus 2 (CoV-2) pandemic. Herein, we establish a simple and sensitive electrochemical assay using magnetic nanocomposite and DNA sandwich probes to rapidly quantify the CoV-2 nucleocapsid (N) gene down to the 0.37 fM level. This assay uses a pair of specific DNA probes. The capture probe is covalently conjugated to Au-decorated magnetic reduced graphene oxide (AMrGO) nanocomposite for efficiently capturing target RNA. In contrast, the detection probe is linked to peroxidase for signal amplification. The probes target the COV-2 gene, allowing for specific magnetic separation, enzymatic signal amplification, and subsequent generation of voltammetric current with a total assay time of 45 min. The developed biosensor has high selectivity and can discriminate non-specific gene sequences. Synthetic COV-2 N-gene can be detected efficiently in serum and saliva, while 1-bp mismatch gene yielded a low response. The performance of the genosensor was good in an extensive linear range of 5 aM–50 pM. For synthetic N-gene, we achieved the detection limit of 0.37, 0.33, and 0.19 fM in human saliva, urine, and serum. This simple, selective, and sensitive genosensor could have various genetics-based biosensing and diagnostic applications.

A SERS bioassay based on vancomycin-modified PEI-interlayered nanocomposite and aptamer-functionalized SERS tags for synchronous detection of Acinetobacter baumannii and Klebsiella pneumoniae

Klebsiella pneumoniae (KP) and Acinetobacter baumannii (AB) are two important gram-negative bacteria that cause pneumonia and have been recently known to be associated with food. The rapid detection of these pathogens in food is important to minimize their colonization of the gut and stop new threats of the disease from spreading across the food chain. Herein, a double-edged sword aptasensor was developed for the synchronous detection of KP and AB in food and clinical samples. A highly sensitive, selective, specific, and synchronous detection of the target bacteria was achieved, and the limit of detection (LOD) was 10 cells/mL with a liner range of 50 to 105 cells/mL. The total assay time was 1.5 h. This study does not only provide a new tool for the detection of the target bacteria, but also serves as a promising tool for food safety and pneumonia diagnosis.

Differential T Cell Responsiveness to Proliferative Stimuli Assessed with a Novel, Homogeneous Ki-67 Immunoassay

Abstract Proliferative capacity is an important component of T cell fitness and a relevant parameter for characterization of CAR T cells and T cell activating agents. Ki-67 protein is a well-validated marker of cell proliferation. We used Lumit™ technology to develop a homogeneous, luminescent immunoassay for determination of intracellular Ki-67 levels. Purified CD8 +T cells from different human donors were plated in 96-well format, then treated with a panel of T cell activators +/− IL-2, including αCD3/αCD28, αCD3/αCD28/αCD2, phytohemagglutinin, concanavalin A, and a cocktail of PMA and ionomycin. An optimized lytic reagent was added to control and treated cells, followed by two anti-Ki-67 monoclonal antibodies labeled with SmBiT and LgBiT subunits, respectively. Upon coincident binding of these labeled antibodies to Ki-67 protein molecules, the resultant proximity of SmBiT and LgBiT reconstitutes NanoBiT luciferase. Addition of Lumit Detection Reagent produces light proportional to the level of Ki-67 expression, with a total assay time of 2 h. It was determined that stimulation of Ki-67 levels varied markedly between T cell activators tested, with the maximum responses observed exceeding 13-fold. Notably, T cell responses varied significantly between donors, including with respect to the degree of IL-2 dependence. This assay system was compatible with same-well fluorescent determination of cell death. In parallel treated wells, Lumit cytokine immunoassays were used to assess concomitant increases in cytokine levels. This simple, no-transfer, no-wash immunoassay for quantitative determination of Ki-67 levels provides an improved workflow for rapid and efficient comparative assessments of T cell proliferative responses.

Simple and effective detection of water in hygroscopic organic solvents using smartphone-based device with dithiophene-conjugated benzothiazole

A dithiophene-bearing benzothiazole derivative (DTBz) was synthesized as a moisture-sensitive fluorescent probe for the detection of water in hygroscopic solvents. In a mixture of water and organic solvents, such as THF, 1,4-dioxane, MeOH, and EtOH, the fluorescence intensity of DTBz showed ratiometric changes in response to the content of water in the organic solvents. Moreover, the emission color of DTBz changed drastically from blue to red, which facilitated the development of the smartphone-assisted fluorometric device using image analysis. The custom smartphone-based device allowed water detection over the range of 0–60% with a resolution of 0.1% in the range of 0–1%. The detection sensitivity was as low as 0.05%. The total assay time was estimated to be approximately 6 min. The custom device would be very useful for determining the water content of various hygroscopic organic solvents over a wide range.