Short Assay Time Research Articles

Glycan-Evocated Metallization for Amplification-Free Electrochemical Detection of Glycoproteins at Low Concentration Levels.

Glycoproteins have become the most often screened tumor markers in the in vitro diagnostics. Although a large number of electrochemical methods have been proposed to sensitively detect glycoproteins, most of them involve the aid of laborious signal amplification. Herein, we report the use of glycan-evocated metallization (GlyMetal) for the amplification-free electrochemical detection of glycoproteins at low concentration levels. Briefly, the glycoproteins of interest are captured by an aptamer recognition layer, and then the glycans of targets are oxidized by NaIO4 to convert the 1,2-diol sites into aldehyde groups for the silver deposition-based metallization, followed by the electrochemical stripping assay of the deposited metallic silver for glycoprotein quantification via the established solid-state Ag/AgCl voltammetric process. As GlyMetal can enable the deposition of a large amount of metallic silver and a high signal-to-background ratio can be obtained for the solid-state Ag/AgCl voltammetric stripping assay, the developed GlyMetal-based electrochemical method is applicable to the amplification-free detection of glycoproteins. As a proof of concept, a detection limit of 1.65 pg/mL has been achieved for carcinoembryonic antigen (CEA) detection. In addition to the high selectivity, desirable results have been obtained with respect to the use of the method for CEA detection in serum samples. In consideration of the desirable simplicity, short assay time, and cost-effectiveness of the amplification-free approach, the GlyMetal-based electrochemical method shows great promise in the point-of-care detection of glycoproteins.

Intraoperative assessment of microimplantation-induced acute brain inflammation with titanium oxynitride-based plasmonic biosensor

Implantable devices for brain-machine interfaces and managing neurological disorders have experienced rapid growth in recent years. Although functional implants offer significant benefits, issues related to transient trauma and long-term biocompatibility and safety are of significant concern. Acute inflammatory reaction in the brain tissue caused by microimplants is known to be an issue but remains poorly studied. This study presents the use of titanium oxynitride (TiNO) nanofilm with defined surface plasmon resonance (SPR) properties for point-of-care characterizing of acute inflammatory responses during robot-controlled micro-neuro-implantation. By leveraging surface-enriched oxynitride, TiNO nanofilms can be biomolecular-functionalized through silanization. This label-free TiNO-SPR biosensor exhibits a high sensitivity toward the inflammatory cytokine interleukin-6 with a detection limit down to 6.3 fg ml−1 and a short assay time of 25 min. Additionally, intraoperative monitoring of acute inflammatory responses during microelectrode implantation in the mice brain has been accomplished using the TiNO-SPR biosensors. Through intraoperative cerebrospinal fluid sampling and point-of-care plasmonic biosensing, the rhythm of acute inflammatory responses induced by the robot-controlled brain microelectrodes implantation has been successfully depicted, offering insights into intraoperative safety assessment of invasive brain-machine interfaces.

Neural Network Enables High Accuracy for Hepatitis B Surface Antigen Detection with a Plasmonic Platform.

The detection of hepatitis B surface antigen (HBsAg) is critical in diagnosing hepatitis B virus (HBV) infection. However, existing clinical detection technologies inevitably cause certain inaccuracies, leading to delayed or unwarranted treatment. Here, we introduce a label-free plasmonic biosensing method based on the thickness-sensitive plasmonic coupling, combined with supervised deep learning (DL) using neural networks. The strategy of utilizing neural networks to process output data can reduce the limit of detection (LOD) of the sensor and significantly improve the accuracy (from 93.1%-97.4% to 99%-99.6%). Compared with widely used emerging clinical technologies, our platform achieves accurate decisions with higher sensitivity in a short assay time (∼30 min). The integration of DL models considerably simplifies the readout procedure, resulting in a substantial decrease in processing time. Our findings offer a promising avenue for developing high-precision molecular detection tools for point-of-care (POC) applications.

Construction of a Spatial-Confined Self-Stacking Catalytic Circuit for Rapid and Sensitive Imaging of Piwi-Interacting RNA in Living Cells.

Piwi-interacting RNAs (piRNAs) are small noncoding RNAs that repress transposable elements to maintain genome integrity. The canonical catalytic hairpin assembly (CHA) circuit relies on random collisions of free-diffused reactant probes, which substantially slow down reaction efficiency and kinetics. Herein, we demonstrate the construction of a spatial-confined self-stacking catalytic circuit for rapid and sensitive imaging of piRNA in living cells based on intramolecular and intermolecular hybridization-accelerated CHA. We rationally design a 3WJ probe that not only accelerates the reaction kinetics by increasing the local concentration of reactant probes but also eliminates background signal leakage caused by cross-entanglement of preassembled probes. This strategy achieves high sensitivity and good specificity with shortened assay time. It can quantify intracellular piRNA expression at a single-cell level, discriminate piRNA expression in tissues of breast cancer patients and healthy persons, and in situ image piRNA in living cells, offering a new approach for early diagnosis and postoperative monitoring.

A dual-channel electrochemical biosensor enables concurrent detection of pathogens and antibiotic resistance

Diarrheagenic E. coli infections, commonly treated with β-lactam antibiotics, contribute to antibiotic resistance - a pressing public health concern. Rapid monitoring of pathogen antibiotic resistance is vital to combat antimicrobial spread. Current bacterial diagnosis methods identify pathogens or determine antibiotic resistance separately, necessitating multiple assays. There is an urgent need for tools that simultaneously identify infectious agents and their antibiotic resistance at the point of care (POC). We developed an integrated electrochemical chip-based biosensor for detecting enteropathogenic E. coli (EPEC), a major neonatal diarrheal pathogen, using an antibody against a virulence marker, termed EspB, and the β-lactam resistance marker, β-lactamase. A dual-channel microfabricated chip, bio-functionalized with a specific EspB monoclonal antibody, and nitrocefin, a β -lactamase substrate was utilized. The chip facilitated electrochemical impedance spectroscopy (EIS)-based detection of EspB antigen and EspB-expressing bacteria. For β-lactam resistance profiling, a second channel enabled differential-pulse voltammetric (DPV) measurement of hydrolyzed nitrocefin. EIS-based detection of EspB antigen was calibrated (LOD: 4.3 ng/mL ±1 and LOQ: 13.0 ng/mL ±3) as well as DPV-based detection of the antibiotic resistance marker, β-lactamase (LOD: 3.6 ng/mL ±1.65 and LOQ: 10 ng/mL ±4). The integrated EIS and DPV biosensor was employed for the simultaneous detection of EspB-expressing and β-lactamase-producing bacteria. The combined readout from both channels allowed the distinction between antibiotic-resistant and -sensitive pathogenic bacteria. The integrated electrochemical biosensor successfully achieved simultaneous, rapid detection of double positive EspB- and β-lactamase-expressing bacteria. Such distinction enabled by a portable device within a short assay time and a simplified sample preparation, may be highly valuable in mitigating the spread of AMR. This new diagnostic tool holds promise for the development of POC devices in clinical diagnosis.

Abstract 3249: Simple, rapid, and robust bioluminescent cell-based assay for detecting neutralizing antibodies against AAV in serum

Abstract Adeno-associated viruses (AAV) are prevalent gene therapy vectors, delivering therapeutic transgenes to treat various diseases including cancer and rare monogenic diseases. Individuals with pre-existing immunity to AAVs are less likely to benefit from AAV-based therapies due to both, a reduction in cellular AAV uptake and activation of a cytotoxic T-cell response to transduced cells, decreasing efficacy or changing the safety profile. Measurement of serum neutralizing antibodies (NAbs) against AAV is essential to determine treatment eligibility and to identify clinical trial candidates. Here we introduce a cell-based assay using NanoLuc® luciferase to detect NAbs in serum samples. The superior brightness of NanoLuc® luciferase in our AAV reporter system results in the need of low concentrations of AAV and a short 24h assay time. The minimal AAV concentration in our assay aids in detecting low NAb titers in serum samples, reducing false negatives and enhancing decision-making accuracy. Testing 60 human serum samples against a range of AAV serotypes, we found that a large fraction of the population had pre-existing NAbs to at least one serotype, and of the pre-exposed individuals a majority displayed NAbs against multiple AAV serotypes. Interestingly, fewer samples were seropositive for AAV-DJ than for either parental serotype (AAV2, AAV8 or AAV9), highlighting the assays utility in the development of engineered capsids. Furthermore, the broad dynamic range of our assay facilitates serum sample categorization into four NAb tiers: negative, low, medium, and high using a single serum dilution. Neutralizing titers (ND50) which was determined by serial dilution of the serum samples strongly agreed with the assigned NAb tiers. Our study with 40 naïve mice further validated the assay's robustness, showing a clear correlation between NAb levels and injected AAV viral loads. Taken together, this rapid, highly sensitive, and reliable assay enabled by NanoLuc® AAV reporter technology precisely measures NAbs against AAVs in both human and animal serum. Further validation of the assay is required for future use in a clinical setting. Citation Format: Morten Seirup, Kirsten Wingate, Jeff Nelson, Kara Machleidt, Vanessa Ott, Trish Hoang, Marjeta Urh. Simple, rapid, and robust bioluminescent cell-based assay for detecting neutralizing antibodies against AAV in serum [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3249.

Enhancing Loop-Mediated Isothermal Amplification through Nonpowered Nanoelectric Preconcentration.

The global threat posed by the COVID-19 pandemic has catalyzed the development of point-of-care (POC) molecular diagnostics. While loop-mediated isothermal amplification (LAMP) stands out as a promising technique among FDA-approved methods, it is occasionally susceptible to a high risk of false positives due to nonspecific amplification of a primer dimer. In this work, we report an enhancing LAMP technique in terms of assay sensitivity and reliability through streamlined integration with a nonpowered nanoelectric preconcentration (NPP). The NPP, serving as a sample preparation tool, enriched the virus concentration in samples prior to the subsequent LAMP assay. This enrichment enabled not only to achieve more sensitive assay but also to shorten the assay time for all tested clinical samples by ∼10 min compared to the conventional LAMP. The shortened assay time suppresses the occurrence of nonspecific amplification by not providing the necessary incubation time, effectively suppressing misidentification by false positives. Utilizing this technique, we also developed a prototype of the POC NPP-LAMP kit. This kit offers a streamlined diagnostic process for nontrained individuals, from the sample enrichment, transfer of the enriched sample to LAMP assays, which facilitates on-site/on-demand diagnosis of SARS-CoV-2. This development holds the potential to contribute toward preventing not only the current outbreak but also future occurrences of pandemic viruses.

Single-Impact Electrochemistry for Bacterial Sensing

The main objective in the detection and analysis of bio-entities such as bacteria is a low detection limit, a short assay time and a high selectivity in identification, ideally combined with easy-handling and cheap instrumentation. In this way, the development of innovative analytical methods in terms of high sensitivity and spatial-temporal resolution allowing both qualitative and quantitative analysis at single-cell and subcellular levels is becoming a technological requirement. The main advantage of electrochemistry in biosensing applications is to combine high sensitivity and ease of use with the possibility of miniaturization at low cost. Especially, new electrochemical techniques and various types of electrodes have been recently developed offering the feasibility of analysis at the single-cell level with high sensitivity and selectivity. Nano-electrochemistry has been widely extended to various applications such as biosensing thanks to the continuous improvement of the instrumentation sensitivity, especially for individual entity electrochemical detection. Single-impact electrochemistry provides a low limit of detection (in principle, one single species) inherent to this electro-analytical method and the ability to study single entities (cells, viruses, bacteria, nanoparticles...) in real-time through a dynamic measurement [1]. The electrochemical nano-impacts method or discrete collisions technique (stochastic events) consists in detecting single impacts of various entities such as nanoparticles, cells, bacteria, vesicles, viruses, proteins in solution at a polarized ultramicroelectrode (UME) [2]. For each collision event, a specific signal is recorded in the chronoamperometry curve (current as a function of time) corresponding to an “impact” of the entity onto the UME surface. Crucial information can be reached from the analysis of the electrochemical impact event in the amperometry measurement, such as the concentration and the size of the colliding entity.Electrochemical nano-impacts (or collisions) of single bacteria are usually detected by recording a chronoamperometry measurement (i–t curve) at a UME biased at a redox potential of an electroactive species in an aqueous electrolyte containing between 106 and 109 bacteria per milliliter [1, 3]. Since 2015, most of the reports in this area concern Escherichia coli (E. coli) as a model bacterium but this sensitive electro-analytical technique has not proved to be efficient for selective detection yet, except for the discrimination between Gram-positive and Gram-negative bacteria. In order to develop single-impact electrochemistry toward the identification of bacterial cells, several studies deal with the use of redox mediators for probing the electrochemical activity of the bacterium. Also, an original and recent approach is to detect the virulence factors released by pathogenic bacteria rather than the cells themselves, based on redox liposomes single-impact electrochemistry [2, 4].

Application of multiple binding sites for LAMP primers across P. falciparum genome improves detection of the parasite from whole blood samples

IntroductionMalaria remains a significant health concern, particularly in regions with widespread prevalence. As the transmission rates decrease, there is a rise in low-density infections with the causative parasite, P. falciparum, that often escape detection through standard point-of-care diagnostic tools. In-low transmission areas, even few undetected cases can trigger outbreaks, necessitating rapid and sensitive diagnostics. Loop-mediated isothermal Amplification (LAMP) stands out as a nucleic acid technique that can easily utilizes un-processed samples such of saliva, urine, and lysed whole blood templates for a sensitive detection. However, most nucleic acid tests detect genes with few copies per parasite making it difficult to detect low-density parasitaemia.MethodsWe selected Pfr364 multi-copy repeats of the P. falciparum genome as a target for amplification due to their higher copy number, ideal for rapid amplification, addressing amplification drawbacks of limited parasites DNA. We used a sequence clustering approach to design a novel set of LAMP primers, capable of binding to multiple sites. Subsequently, we developed a hydroxynaphthol blue (HNB) colorimetric LAMP assay, using genomic DNA obtained from the 3D7 strain cultivated in vitro. This assay’s performance was validated using archived clinical samples of both whole blood and matched saliva, ensuring accuracy through comparative analysis against gold standard, nested PCR, targeting the 18S RNA gene.ResultsThe HNB-LAMP assay achieved rapid amplification within 15 minutes and exhibited high sensitivity with a limit of detection of 1 parasite. Further, the LAMP assay was robust in whole blood lysed with Triton X-100 and heat-treated saliva clinical samples. Against nested PCR, the assay showed sensitivity of 100% for whole blood and 40% for saliva samples. Moreover, co-analysis with the nested PCR showed a perfect agreement between the two techniques. (K = 0.99 for whole blood, and 0.66 for saliva).ConclusionOur study presents a method for detecting P. falciparum using LAMP, which results in increased sensitivity, shorter assay times, and a simpler workflow than nucleic acid tests relying on conventional DNA extraction and additional equipment for result interpretation. These findings hold great promise for improved malaria diagnosis, especially in settings where low-density parasitaemia is prevalent and rapid and accurate malaria detection is crucial.

Development of a receptor based signal amplified fluorescence polarization assay for multi-detection of 35 sulfonamides in pork.

With the increasing focus on food security, a screening method with high-throughput, ultra-sensitivity, and user-friendly operation is urgently needed for monitoring of sulfonamides residues in animal-derived foods. In this study, the sulfonamides' receptor dihydropteroate synthase of Staphylococcus aureus was subjected to saturate mutation, and a mutant with higher affinities for sulfonamides was obtained. The mutant was then used as recognition material to establish a fluorescence polarization assay for determination of 35 sulfonamides in pork. Due to the use of an enhanced fluorescent tracer containing two fluorophore molecules, the sensitivities for the 35 sulfonamides were improved for 2.8-8.6 folds (LODs 0.03-1.16ng/mL) in comparison with using conventional fluorescent tracer.The present method outperformed all previous fluorescence polarization (immuno)assays for sulfonamides due to its broader spectrum, higher sensitivity, and shorter assay time. Furthermore, this is the first study reporting an enhanced fluorescence polarization assay for determination of small molecule substance.

Evaluation of an In-house Indirect ELISA for Differential Detection of IgM and IgG anti-Brucella Antibodies in Human Brucellosis

Brucellosis caused by various species of the genus Brucella is one of the most important zoonotic diseases of global importance with veterinary, public health, and economic concerns. The study aimed to standardize IgM and IgG-based iELISA to detect anti-Brucella antibodies for serodiagnosis of acute and chronic human brucellosis. The test was standardized using 1:320 dilution of smooth lipopolysaccharide (sLPS) antigen from B. abortus S99 strain, 1:80 serum dilution, 1:4000 anti-human IgM and IgG conjugates, respectively for both IgM and IgG iELISA. The cut-off using 50 each brucellosis positive and negative human sera panel samples was set at ≥ 42 for both IgM and IgG iELISA. A total of 700 human sera samples were evaluated (137 veterinary doctors, 157 artificial inseminators, and 406 veterinary assistants). Overall, the study detected 8.3%, 8.1%, 8%, and 6.1% positivity by in-house IgG iELISA, RBPT, IgM iELISA, and SAT tests, respectively. Considering commercial iELISA kit as a gold standard, the sensitivities of IgM and IgG iELISA were 90% and 97.9%, respectively, whereas, specificities were >99%. The study established >98% specificity and >90% sensitivity for differential detection of immunoglobulin classes in the standardized iELISA. The developed assay outperformed the other evaluated tests with a shorter assay time and can be implemented in both endemic and non-endemic regions for surveillance and diagnosis of human brucellosis.

Marker-Free Isoelectric Focusing Patterns for Identification of Meat Samples via Deep Learning.

Isoelectric focusing (IEF) is a powerful tool for resolving complex protein samples, which generates IEF patterns consisting of multiplex analyte bands. However, the interpretation of IEF patterns requires the careful selection of isoelectric point (pI) markers for profiling the pH gradient and a trivial process of pI labeling, resulting in low IEF efficiency. Here, we for the first time proposed a marker-free IEF method for the efficient and accurate classification of IEF patterns by using a convolutional neural network (CNN) model. To verify our method, we identified 21 meat samples whose IEF patterns comprised different bands of meat hemoglobin, myoglobin, and their oxygen-binding variants but no pI marker. Thanks to the high throughput and short assay time of the microstrip IEF, we efficiently collected 1449 IEF patterns to construct the data set for model training. Despite the absence of pI markers, we experimentally introduced the severe pH gradient drift into 189 IEF patterns in the data set, thereby omitting the need for profiling the pH gradient. To enhance the model robustness, we further employed data augmentation during the model training to mimic pH gradient drift. With the advantages of simple preprocessing, a rapid inference of 50 ms, and a high accuracy of 97.1%, the CNN model outperformed the traditional algorithm for simultaneously identifying meat species and cuts of meat of 105 IEF patterns, suggesting its great potential of being combined with microstrip IEF for large-scale IEF analyses of complicated protein samples.

Rapid and label-free electrochemical aptasensor based on a palladium nanoparticles/titanium carbide/polyethyleneimine functionalized nitrogen-doped carbon nanotubes composite for amplified detection of streptomycin

A novel electrochemical aptasensor for the determination of streptomycin (STR) was constructed in this work. We prepared a palladium nanoparticles/titanium carbide/polyethyleneimine–functionalized nitrogen-doped carbon nanotube (Pd@Ti3C2-PEI-NCNTs) composite via hydrothermal-assisted formic acid reduction and employed it as a substrate material for anchoring a NH2-functionalized aptamer (Apt) to the surface of an electrode through the Pd-N bond. The Pd@Ti3C2-PEI-NCNTs composite increased the electrochemically active surface area and conductivity of the electrode, achieving substantial signal amplification (1.87 times). The constructed aptasensor demonstrated accurate STR detection via specific recognition between the Apt and STR, with a detection range and limit of 0.01–700 and 0.003 nmol/L, respectively. The aptasensor also exhibited excellent selectivity, stability, reproducibility, practicability and shorter assay times (60 min). Furthermore, molecular docking and molecular dynamic also elucidated the binding mechanism between the aptamer and STR molecules. Overall, this method is expected to be a helpful tool for detecting STR in food.

Modular DNAzymes-Hydrogel Membrane Carriers for Highly Sensitive Isothermal Cross-Cascade Detection of Pathogenic Bacteria Nucleic Acids.

The increasing prevalence of antimicrobial resistance has called for improved diagnostic testing of pathogenic bacteria. However, the development of rapid, cost-effective, and easy-to-use tests for bacterial infections remains a constant challenge. Here, we report a class of modular hydrogel membrane carriers incorporated with composite DNAzymes, which enable rapid and highly sensitive detection of pathogenic bacteria gene target analytes. We apply free radical polymerization to incorporate composite DNAzymes, consisting of an RNA substrate component and a DNAzyme component (e.g., 10-23 or 8-17 DNAzymes), into polyethylene glycol diacrylate polymer networks. Initiated by a nucleic acid target acting as an assembly facilitator, multicomponent DNAzymes are combined to cleave the RNA substrate component in the hydrogel carriers, which releases the DNAzyme component to cleave RNA reporter probes to generate fluorescence. We modulate the morphology, composition, and microporous structures of the DNAzyme carriers to achieve quantitative assay performance. We demonstrate a rapid and high-sensitivity detection of C. trachomatis gene target analytes as low as 50 fM in a short assay time of 25 min. The work represents a crucial step forward in the development of a generic, isothermal, and protein enzyme-free pathogenic bacteria testing platform technology.

One-step and real-time detection of Hg2+ in brown rice flour using a biosensor integrated with AC electrothermal enrichment.

Mercury (Hg2+) is one of the most toxic heavy metals in farm products, so rapid detection of trace Hg2+ has always been sought after with high interest. Herein, we report a biosensor to specifically recognize Hg2+ in leaching solutions of brown rice flour. This sensor is simple and of low cost, with a very short assay time of 30s. Another merit is the ultra-low limit of detection (LOD) at fM level. In addition, the specific aptamer probe realizes a good selectivity above 105: 1 against the interferences. This sensor is developed based on an aptamer-modified gold electrode array (GEA) for capacitive sensing. Alternating current electrothermal (ACET) enrichment is induced during the AC capacitance acquirement. Thus, the enrichment and detection are coupled as a single step, and pre-concentration is needless. Owing to the sensing mechanism of solid-liquid interfacial capacitance and ACET enrichment, Hg2+ level can be sensitively and rapidly reflected. Also, the sensor has a wide linear range from 1 fM to 0.1nM and a shelf life of 15days. This biosensor shows advantages on overall performance, enabling easy-to-operate, real-time, and large-scale Hg2+ detection in farm products.

Silica nanoparticle-modified paper strip-based new rhodamine B chemosensor for highly selective detection of copper ions in drinking water.

A new rhodamine B derivative (RDB) was synthesized and utilized for the colorimetric detection of copper ions (Cu2+). This chemosensor utilized a paper strip as a support and a smartphone as a detector for on-site quantitative detection of Cu2+ in water samples. Silica nanoparticles (SiNPs) were investigated as the modifier nanoparticles to achieve uniform color on the paper strip and showed a color response 1.9-fold higher than the one without SiNPs. The RDB chemosensor-based paper strip provided high selectivity toward Cu2+ with a detection limit of 0.7mg/L, and the working concentrations for Cu2+ ranged from 1 to 17mg/L. Parallel analyses of eight drinking water samples were conducted by inductively coupled plasma optical emission spectroscopy. The results were in good agreement, indicating the practical reliability of the established method with a short assay time and high selectivity. These indicate its great potential for on-site detection of Cu2+.

Immediate, sensitive and specific time-resolved fluorescent immunoassay strips based on immune competition for the detection of procymidone in vegetables

As an organochlorine pesticide, procymidone has become an increasing serious problem in vegetables in recent years. In order to solve a series of problems such as low sensitivity of the immunological methods, a time-resolved fluorescent immunoassay strip (TRFIAS) was developed with low limit of detection (LOD). Based on the principle of antigen-antibody immune competition, TRFIAS could detect procymidone in vegetables. After the targets being added, the samples were incubated at 37 °C for 10 min. TRFIAS was observed under a UV lamp of 365 nm. The results were processed by ImageJ software to complete the qualitative and quantitative analysis of TRFIAS. Under the optimal working conditions, a linear range of TRFIAS was 5–250 ng/mL with a LOD of 0.33 ng/mL and a visual limit of detection (VDL) of 50 ng/mL. And the TRFIAS had good specificity, precision and accuracy. Comparing the results of TRFIAS with those of ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), the two assays were in good agreement. Therefore, TRFIAS has the advantages of high sensitivity, high specificity, simple operation and relatively short assay time. Meanwhile, it shows absolutely great advantages in both quantitative and qualitative analysis.

Recent Applications and Future Perspectives of Chemiluminescent and Bioluminescent Imaging Technologies.

Imaging technologies based on chemiluminescence (CL) and bioluminescence (BL) have seen a tremendous growth in the past decade due to their extensive contributions to biochemical analysis and biomedical science. CL and BL imaging technologies have many advantages over commonly used imaging techniques (such as fluorescence and electrochemical systems). CL and BL proceed without an external light source, thus avoiding photobleaching, high interference of background signal, and autofluorescence. Furthermore, CL and BL analytical techniques are characterized by their low detection limit, high selectivity, short assay time, and simple instrumentation. Recently, CL-based imaging technology has been applied successfully for the determination and in vivo imaging of several significant analytes. Meanwhile, an innovative BL imaging technology has been established for the noninvasive real-time tracking of different biomolecules relying on the interaction between luciferase and its substrate. In the current review, we discuss the recent applications of CL and BL imaging approaches over the past five years. Finally, we also discuss the current state of progress and the prospects for CL and BL imaging systems.

Rapid and Visual Detection of Oxytetracycline Using Mesoporous Silica Nanochannels Modified Interdigital Electrode

Oxytetracycline (OTC) is one of the widely used antibiotics in veterinary practices because of its broad-spectrum antibacterial activity and low cost. However, drug abuse has triggered adverse effects on human health, which brings a growing demand for on-site diagnosis of OTC residues in animal-derived foods. In this study, we demonstrated the combined use of interdigital electrode microarray and electrochemiluminescence (ECL) imaging for parallel multiplex measurement of OTC. Well-ordered and vertically aligned mesoporous silica nanochannels modified on microarray substrate could exert a strong electrostatic attraction to the ECL luminophores and accelerate their mass transport to generate enhanced ECL signal. The performance of the integrated ECL microarray sensor was fully validated with respect to linearity (0.5 μM to 50 μM), sensitivity (limit of detection 0.26 μM), accuracy (recovery rate between 96.78% and 106.1%), low operating sample volume (480 nL), short assay time (1.5 min), and antifouling ability toward complex media. The multiplex microarray platform can serve as a simple, fast, and low-cost tool for the detection of a wide spectrum of antibiotics in the field of food safety.

A digital nanoplasmonic microarray immunosensor for multiplexed cytokine monitoring during CAR T-cell therapy from a leukemia tumor microenvironment model

The release of cytokines by chimeric antigen receptor (CAR) T-cells and tumor resident immune cells defines a significant part of CAR T-cell functional activity and patient immune responses during CAR T-cell therapy. However, few studies have so far precisely characterized the cytokine secretion dynamics in the tumor niche during CAR T-cell therapy, which requires multiplexed, and timely biosensing platforms and integration with biomimetic tumor microenvironment. Herein, we implemented a digital nanoplasmonic microarray immunosensor with a microfluidic biomimetic Leukemia-on-a-Chip model to monitor cytokine secretion dynamics during CD19 CAR T-cell therapy against precursor B-cell acute lymphocytic leukemia (B-ALL). The integrated nanoplasmonic biosensors achieved precise multiplexed cytokine measurements with low operating sample volume, short assay time, heightened sensitivity, and negligible sensor crosstalk. Using the digital nanoplasmonic biosensing approach, we measured the concentrations of six cytokines (TNF-α, IFN-γ, MCP-1, GM-CSF, IL-1β, and IL-6) during first 5 days of CAR T-cell treatment in the microfluidic Leukemia-on-a-Chip model. Our results revealed a heterogeneous secretion profile of various cytokines during CAR T-cell therapy and confirmed a correlation between the cytokine secretion profile and the CAR T-cell cytotoxic activity. The capability to monitor immune cell cytokine secretion dynamics in a biomimetic tumor microenvironment could further help in study of cytokine release syndrome during CAR T-cell therapy and in development of more efficient and safer immunotherapies.