Bioanalytical Applications Research Articles

Spatially resolved metabolomics: From metabolite mapping to function visualising.

Mass spectrometry imaging (MSI)-based spatially resolved metabolomics addresses the limitations inherent in traditional liquid chromatography-tandem mass spectrometry (LC-MS)-based metabolomics, particularly the loss of spatial context within heterogeneous tissues. MSI not only enhances our understanding of disease aetiology but also aids in the identification of biomarkers and the assessment of drug toxicity and therapeutic efficacy by converting invisible metabolites and biological networks into visually rendered image data. In this comprehensive review, we illuminate the key advancements in MSI-driven spatially resolved metabolomics over the past few years. We first outline recent innovations in preprocessing methodologies and MSI instrumentation that improve the sensitivity and comprehensiveness of metabolite detection. We then delve into the progress made in functional visualization techniques, which enhance the precision of metabolite identification and annotation. Ultimately, we discuss the significant potential applications of spatially resolved metabolomics technology in translational medicine and drug development, offering new perspectives for future research and clinical translation. HIGHLIGHTS: MSI-driven spatial metabolomics preserves metabolite spatial information, enhancing disease analysis and biomarker discovery. Advances in MSI technology improve detection sensitivity and accuracy, expanding bioanalytical applications. Enhanced visualization techniques refine metabolite identification and spatial distribution analysis. Integration of MSI with AI promises to advance precision medicine and accelerate drug development.

Rapid Thermal Drying Synthesis of Nonthiolated Spherical Nucleic Acids with Stability Rivaling Thiolated DNA

AbstractAttaching DNA oligonucleotides to gold nanoparticles (AuNPs) to prepare spherical nucleic acids (SNAs) has offered tremendous insights into surface chemistry with resulting bioconjugates serving as critical reagents in biosensors and nanotechnology. While thiolated DNA is generally required to achieve stable conjugates, we herein communicate that using a thermal drying method, a high DNA density and excellent SNA stability was achieved using nonthiolated DNA, rivaling the performance of thiolated DNA such as surviving 1 M NaCl, 2 month stability in 0.3 M NaCl and working in 50 % serum. A poly‐adenine block with as few as two consecutive terminal adenine bases is sufficient for anchoring on AuNPs. By side‐by‐side comparison with the salt‐aging method, the conjugation mechanism was attributed to competitive adenine adsorption at high temperature along with an extremely high DNA concentration upon drying. Bioanalytical applications of nonthiolated SNAs were validated in both solution and paper‐based sensor platforms, facilitating cost‐effective applications for SNAs.

Probing the role of ligation and exonuclease digestion towards non-specific amplification in bioanalytical RCA assays.

Non-specific amplification (NSA, amplification in the absence of a target analyte) in bioanalytical rolling circle amplification (RCA) assays, especially those involving pre-synthesized circular DNA (cDNA), affects its analytical sensitivity. Despite extensive development of RCA-based bioanalytical methods, the NSA in RCA remains uncharacterized in terms of its magnitude or origin. NSA may originate from inefficient ligation or succeeding cDNA purification steps. This study comprehensively quantifies NSA across several ligation and digestion techniques for the first time since the innovation of RCA. To quantify the NSA in RCA, cDNAs were prepared using self-annealing, splint-padlock, or cohesive end ligations. The cDNAs were then subjected to nine different exonuclease digestion steps and quantified for NSA under linear as well as hyperbranched RCA conditions. We investigated buffer compositions, divalent ion concentrations, single or dual enzyme digestion, cohesive end lengths, and splint lengths. The optimized conditions successfully mitigated absolute NSA by 30-100-fold and relative NSA (normalized against primer-assisted RCA) to ∼5%. Besides understanding the mechanistic origin of NSA, novel aspects of enzyme-substrate selectivity, buffer composition, and the role of divalent ions were discovered. With increasing bioanalytical RCA applications, this study will help standardize NSA-free assays.

A novel CRISPR/Cas13a biosensing platform comprising dual hairpin probe and traditional lateral flow assays

Recently, CRISPR/Cas13a-based biosensors have been combined with non-traditional lateral flow assays (N-TLFAs) successfully used to clinical detection of target RNA due to their simplicity, portability, and cost-effectiveness. However, N-TLFAs used in such biosensors have several disadvantages, such as inversion of test and control zones, in which color intensity depends on reporter probe cleavage. Herein, we propose a novel biosensor in which CRISPR/Cas13a is integrated with traditional lateral flow assays (TLFAs) to facilitate the sequential direct location of test/control zones by dual hairpin probes, namely DNA hairpin reporter probe B-HPs (5′ Biotin-hairpins, B-HPs, loop containing 5 rU) and F-HPs (5′ FAM-hairpins, F-HPs). Cas13a activated by crRNA-targeted RNA duplexes can indiscriminately cleave B-HPs reporter probes to release short sequences to open hairpin F-HPs reporter probes to form Biotin-dsDNA-FAM reporter probes. The advantage of this platform is that detection results only rely on the formed Biotin-dsDNA-FAM reporter probes, which can be used for SARS-CoV-2 RNA detection after preliminary detection. Under optimized conditions, the SARS-CoV-2 RNA detection range was 10 aM to 1 µM and the limit of detection was as low as 5.34 aM. This proof-of-concept study demonstrates that dual-hairpin reporter-probe binding to TLFAs opens up new opportunities for CRISPR/Cas13a activity detection and bioanalytical applications.

Digestion-Free Middle-Down Mass Spectrometry Method for Absolute Quantification of Conjugated Payload from Antibody-Drug Conjugates.

Antibody-drug conjugate (ADC) is a therapeutic modality that aims to improve payload delivery specificity and reduce systemic toxicity. Considering the complex structure of ADCs, various bioanalytical methods by liquid chromatography coupled with mass spectrometry (LC-MS), ligand binding assay (LBA) and hybrid LBA-LC-MS approaches have been established for ADC characterization and quantification. LCMS-based assays enable drug-antibody ratio (DAR) sensitive quantification of the conjugated payload. Typically, for quantitative, DAR-sensitive, assessment by LC-MS/MS,the conjugated payload is enzymatically liberated and quantified. Despite recent advances in ADC bioanalytical methods, the DAR-sensitive quantification of noncleavable linker ADCs by LC-MS/MS remains challenging. Thus, we developed a novel digestion-free middle-down mass spectrometry (DF-MDMS) using a collision-induced dissociation approach for absolute quantification of conjugated payload from four different ADCs in a biological matrix with minimum sample preparation. These results demonstrate that ADCs with different linker-payload structures can be quantified, including a noncleavable linker ADC, trastuzumab emtansine. It also shows that the assay sensitivity is comparable to the conventional ADC quantification method by linker-payload cleavage using enzyme, while the assay dynamic range depends on factors including payload ionization and dissociation efficiency, DAR and its distribution, and species abundance. By demonstrating absolute quantification of both cleavable and noncleavable linker ADCs, this novel middle-down ADC approach demonstrates its potential application in bioanalysis and analytical characterization, especially for early discovery where high-throughput screening is required as the new approach saves time and resources by not requiring enzymatic digestion for cleavable ADCs or development of anti-payload antibodies for noncleavable linker ADCs.

Multifunctional Ethyl Violet@NH2-MIL-88B(Fe) Hybrids: CRISPR-Cas12a-Assisted PEC-FL-CL Triple-Mode Sensitive Detection of HPV-16.

The multimode assay based on multiple response mechanisms has received great attention to effectively improve the accuracy of a sensing platform. However, multifunctional sensing materials for simultaneously satisfying the multiple-mode detections are still in shortage due to the incompatibility of the signal transduction mechanisms in different modes. Here, taking human papillomavirus 16 (HPV-16) DNA (TDNA) as the model due to its important role in cervical cancer, a novel multifunctional material, ethyl violet (EV)@NH2-MIL-88B(Fe) (ENM) hybrids, have been successfully prepared, which could simultaneously satisfy CRISPR-Cas12a-assisted photoelectrochemical (PEC)-fluorescent (FL)-colorimetric (CL) triple-mode detection of TDNA. Based on the TDNA-induced trans-cleavage ability of CRISPR-Cas12a and efficient separation of magnetic beads, ENM was obtained from the single-stranded DNA-surrounded streptavidin-modified magnetic beads-ENM (SMB-ssDNA-ENM) and decomposed by pyrophosphate to get free EV, 2-aminoterephthalic acid (NH2-BDC), and Fe3+. Thus, TDNA was sensitively detected based on the EV-enhanced PEC signal of SnS2 nanosheets (PEC mode), fluorescent signal of NH2-BDC (FL mode), and characteristic absorption peak at about 720 nm of Fe3+-induced Prussian blue (PB) (CL mode). The designed PEC-FL-CL triple-mode biosensing platform had good performance for the detection of TDNA with a wide linear range (0.1 fM-100 nM) and ultralow detection limits (0.07 fM for PEC, 0.03 fM for FL and 0.09 fM for CL). Additionally, the developed PEC-FL-CL triple-mode biosensing platform has great potential for applications in early disease diagnosis and bioanalysis, as it can be easily extended to other DNA assays through modification of the crRNA sequence within the CRISPR-Cas12a system.

Capillary-Assisted Assembly of Polymer Gel-Supported Lipid Bilayers.

The polymer-supported lipid bilayer represents an attractive supramolecular assembly in numerous biophysical and bioanalytical applications. The assembly of polymer-supported membranes with a polymer layer thickness of just a few nanometers is now well-established, but bilayer properties in such a membrane architecture are still influenced by the nearby solid substrate. Polymer-supported lipid bilayer systems with a several micrometers thick polymer layer will overcome this shortcoming. However, formation of a fluid lipid bilayer on a fully hydrated, micrometer thick polymer film using traditional methods (e.g., vesicle fusion and lipid monolayer deposition techniques) remains a challenging task due to the rather unfavorable interfacial conditions for bilayer formation in such a system. Here, we report for the first time on the facile capillary-assisted formation of a lipid bilayer on the surface of a fully hydrated, several micrometers thick polyacrylamide (PAA) gel, in which forced molecular crowding of lipids at the air-water interface of the capillary results in monolayer instability and collapse, thereby forming a lipid bilayer on the top of the polymer gel inside the capillary. Stable bilayer attachment on the surface of the polymer gel can be achieved via physisorption or specific chemical linkages (tethering) on both cross-linked and non-cross-linked PAA films. Unlike the traditional solid-supported lipid bilayer (SLB), the lipid lateral diffusion in the polymer gel-supported lipid bilayer is not anymore perturbed by a solid substrate. Instead, more like a plasma membrane, it is mainly influenced by the properties of the underlying polymer and the nature/distribution of polymer-bilayer attachments. Polymer gel-supported lipid bilayers built using the capillary-assisted assembly approach show attractive self-healing properties, resulting in superior long-term stability relative to the SLB. We hypothesize that the described capillary-assisted assembly method can be applied to a wide range of polymeric materials and lipid compositions, opening exciting opportunities as an advanced model membrane system.

Efficient synthesis of the undiluted vegetable juice-compatible hydrophilic quantum dot-labeled fluorescent molecularly imprinted polymer microspheres via facile one-pot surface-initiated ARGET ATRP

The development of fluorescent molecularly imprinted polymers (MIPs) applicable for herbicide optosensing in the undiluted vegetable juices is highly important for food safety control, but still remains a challenging task. Herein, we report on a facile and efficient new strategy for the synthesis of hydrophilic quantum dot (QD)-labeled fluorescent MIP microspheres capable of directly and selectively optosensing a widely used herbicide (i.e., 2,4-dichlorophenoxyacetic acid, 2,4-D) in the undiluted vegetable juices. Such fluorescent MIP particles were readily prepared through one-step grafting of a red CdTe QD-labeled 2,4-D-MIP layer with hydrophilic polyethylene glycol brushes onto the preformed uniform “living” polymer microspheres (with surface-bound alkyl halide groups) via one-pot surface-initiated activator regenerated by electron transfer (ARGET) atom transfer radical polymerization (ATRP) (SI-ARGET ATRP). They proved to be a highly useful optosensor with high 2,4-D selectivity and sensitivity (the limit of detection ≤ 0.14 μM) in the undiluted Chinese cabbage and cucumber juices. They also showed excellent photostability and reusability and could be directly utilized for 2,4-D detection with high recoveries (98.2 %-102.4 %) and accuracy (RSD≤3.8 %) in the undiluted Chinese cabbage and cucumber juices at three spiking levels of both 2,4-D and its mixtures with several analogues. The use of SI-ARGET ATRP significantly reduced the applied copper catalyst concentration (about 5 μM), which greatly alleviated the quenching effect of the copper catalyst on the fluorescence of the QD-labeled MIP (usually observed for those prepared via normal ATRPs that typically use several hundreds of μM of copper catalysts), thus making this facile new strategy highly efficient for developing advanced fluorescent MIPs that hold much promise in various bioanalytical and diagnostic applications.

Versatile Bovine Serum Albumin as Ingenious Electron Operator-Enhanced Photoelectrochemical Biosensing for Ultrasensitive Detection of miRNA.

Bovine serum albumin (BSA) has been widely used in biosensors as a blocking agent. Herein, conformist BSA was first exploited as an ingenious operator to enhance the photocurrent response of (2Z,2'Z)-2,2'-(1,4-phenylene)bis(3-(4-(bis(4-methoxyphenyl)amino)phenyl)acrylonitrile) (TPDCN)-based photoelectrochemical (PEC) platform via manipulating the electron transfer process of the detection system. Concretely, the presence of target molecules triggered catalytic hairpin assembly reaction and subsequently powered terminal deoxynucleotidyl transferase-mediated signal amplification to produce the AgNP@BSA-DNA dendrimer nanostructure. After being treated with HNO3, a large amount of BSA could be released from the dendrimer nanostructure. When they were transferred to the TPDCN-based PEC platform, the photocurrent response of the biosensor was largely enhanced because BSA can manipulate the electrons of TPDCN via a well-matched energy level to form a new electron transfer track. Meanwhile, tryptophan (Trp) in BSA could be oxidized to quinone Trp-O under photoirradiation, which can facilitate the oxidation of ascorbate and generate more H+ to promote the migration of photogenerated electrons. As a result, the proposed PEC biosensor exhibits excellent analytical performance for detection of miRNA-21 (as a model target) over a wide linear range of 0.01 to 10,000 pM with detection limit as low as 4.7 fM. Overall, this strategy provides a new perspective on constructing efficient PEC biosensors, which expands the potential applications in bioanalysis and clinical diagnosis.

Affinity-Controlled Partitioning of Biomolecules at Aqueous Interfaces and Their Bioanalytic Applications.

All-aqueous phase separation systems play essential roles in bioanalytical and biochemical applications. Compared to conventional oil and organic solvent-based systems, these systems are characterized by their rich bulk and interfacial properties, offering superior biocompatibility. In particular, phase separation in all-aqueous systems facilitates the creation of compartments with specific physicochemical properties, and therefore largely enhances the accessibility of the systems. In addition, the all-aqueous compartments have diverse affinities, with an important property known as partitioning, which can concentrate (bio)molecules toward distinct immiscible phases. This partitioning affinity imparts all-aqueous interfaces with selective permeability, enabling the controlled enrichment of target (bio)molecules. This review introduces the basic principles and applications of partitioning-induced interfacial phenomena in a typical all-aqueous system, namely aqueous two-phase systems (ATPSs); these applications include interfacial chemical reactions, bioprinting, and assembly, as well as bio-sensing and detection. The primary challenges associated with designing all-aqueous phase separation systems and several future directions are also discussed, such as the stabilization of aqueous interfaces, the handling of low-volume samples, and exploration of suitable ATPSs compositions with the efficient protocol.

Rapid Thermal Drying Synthesis of Nonthiolated Spherical Nucleic Acids with Stability Rivaling Thiolated DNA.

Attaching DNA oligonucleotides to gold nanoparticles (AuNPs) to prepare spherical nucleic acids (SNAs) has offered tremendous insights into biointerface chemistry with resulting bioconjugates serving as critical reagents in biosensors and nanotechnology. While thiolated DNA is generally required to achieve stable conjugates, we herein communicate that using a thermal drying method, a high DNA density and excellent SNA stability was achieved using nonthiolated DNA, rivaling the performance of thiolated DNA such as surviving 1 M NaCl, 2 month stability in 0.3 M NaCl and working in 50% serum. A poly-adenine block with as few as two consecutive terminal adenine bases is sufficient for anchoring on AuNPs. By side-by-side comparison with the salt-aging method, the conjugation mechanism was attributed to competitive adenine adsorption at high temperature along with an extremely high DNA concentration upon drying. Bioanalytical applications of the nonthiolated SNAs were validated in both solution and paper-based sensor platforms, facilitating cost-effective applications for SNAs.

Revolutionizing Surface Immobilization Techniques for Extended Gate Electric-Double-Layer (EDL) Bio-FETs

The development of biosensors, known for their high sensitivity and specificity, has sparked high interest in semiconductor-related studies and bioanalysis. Within this domain, the establishment of immobilization techniques has become significant. However, conventional antibody immobilization on gold surface techniques using PEG linkers, Traut’s reagent, and 2-Mercaptoethylamine (2-MEA) have some drawbacks such as long debye length, may interact with other amino groups on the antibody, impacting the antibodies' binding affinity, and half-antibody will reduce antibody activity respectively. In this study, we present a novel surface immobilization strategy by using a short DNA sequence. Through the characteristics of synthetic DNA, we can modify its 3’ and 5’ ends with different functional group, enabling effective immobilization of antibodies across diverse sensor surfaces. By doing so, we can match the specific requirements of various materials and test subjects. Also, we can verify the successful surface immobilization by fluorescent labeling. The length of short DNA sequence is only about 1 nanometer, which is much shorter than PEG Linker (> 4.8 nm), enabling a reduced debye length. Firstly, aptamer was immobilized on the surface as the first layer. This modified aptamer carries a disulfide bond at its 5' end, a fluorescent dye (FAM) at its 3' terminal, and an amino group (-NH2) at its center, as shown as Figure 1(A). The amino group served as a functional linker facilitating the attachment of the antibody onto the gold extended gate, providing a stable linkage. After the surface treatment with ATA, the sensor surface was incubated again with EDC/NHS antibody solution. This particular antibody was labeled using the CF® Dye Antibody Label (ROX) conjugate, which responds to excitation by light wavelengths falling within the range of 580 to 610 nanometers (nm). The distinctive red color serves as an indicator to confirm the successful immobilization of the antibody, as shown as Figure 1(B). The process began with the cleaning of the electrode surface using oxygen plasma. Subsequently, the probe was immobilized onto the cleaned surface. To prepare the ATA aptamer, it was mixed with TCEP for 30 minutes at room temperature. The solution mixture was then applied onto the sensor surface and left to immobilize at 24°C for 24 hours. To eliminate unbound aptamers, the sensor underwent a washing step using 1× PBS before applying the antibody solution. We clearly notice a substantial increase in fluorescent intensity following the implementation of the new strategy, as shown in Figure 1. After checking the immobilization of ATA and antibody, the electrical signal will be tested by dropping different concentrations of target antigen. The system we developed is using N-Channel Enhancement-Mode DMOS FET (VN10LP). For the measurement setting, a constant drain voltage (Vd) was applied at 3.5 V. The signals (changes of drain currents, ΔId) were measured when applying gate biases (Vg) at -2 V and 3 V. A signal was read out every 20 seconds, and 10 measurements were taken for each concentration. Real-time data recorded the signal change under different concentrations of target protein from 0, 0.1, 10, 25, 100 ng/mL, as shown as Figure 1(C). Comparing the fluorescent and electrical outcomes between ATA with a PEG linker and Traut’s reagent allows us to address shortcomings encountered in conventional techniques, offering promising solutions for enhanced specificity, improved uniformity, and minimized interference with antibody binding affinity. The successful integration of this novel immobilization strategy into the EDL-FET detection platform marks a significant stride forward in advancing biosensing technologies, promising heightened accuracy and efficiency in bioanalytical applications. Figure 1

Electron-Accelerator-Induced Fast Electron Transfer for Enhancing Electrochemiluminescence of Gold Nanoclusters and Its Bioanalysis Application: A Novel Avenue for Developing High-Efficient Emitters.

Herein, the gold nanoclusters/CaFe2O4 nanospheres (Au NCs/CaFe2O4) heterostructure as a novel electrochemiluminescence (ECL) emitter was developed. Excitingly, Au NCs/CaFe2O4 displayed highly efficient and greatly stable ECL based on the newly defined electron-accelerator p-type semiconductor CaFe2O4 NS-induced fast electron transfer; it solved one key obstacle of metal NC-based ECL emitters: sluggish through-covalent bond electron transport kinetics-caused inferior ECL performance. Specifically, on account of the energy level matching between emitter Au NCs and electron-accelerator CaFe2O4 NSs, the valence band (VB) of the electron-accelerator could provide abundant holes for rapidly transporting the electrogenerated electron from the highest occupied molecular orbital (HOMO) of Au NCs to the electrode, generating massive excited species of Au NCs for strong ECL emission. Notably, Au NCs/CaFe2O4 emerged 5.4-fold higher ECL efficiency with 3.5-fold higher electrochemical oxidation current in comparison with pure Au NCs, exhibiting great prospects in extensive lighting installations, ultrasensitive biosensing, and high-resolution ECL imagery. As applications, an ECL bioassay platform was constructed with Au NCs/CaFe2O4 as an emitter and U-like structure-fueled catalytic hairpin assembly (U-CHA) as a signal amplifier for fast and trace analysis of aflatoxin B1 (AFB1) with the detection limit (LOD) down to 2.45 fg/mL, which was 3 orders of magnitude higher than that of the previous ECL biosensors with much better stability. This study developed an entirely new avenue for enlarging the ECL performance of metal NCs, and it is a very attractive orientation for directing the reasonable design of prominent metal NC-based ECL emitters and broadening the practical application of metal NCs.

Matrix effects in ultra-high performance supercritical fluid chromatography-mass spectrometry analysis of vitamin E in plasma: The effect of sample preparation and data processing

The approaches to matrix effects determination and reduction in ultra-high performance supercritical fluid chromatography with mass spectrometry detection have been evaluated in this study using different sample preparation methods and investigation of different calibration models. Five sample preparation methods, including protein precipitation, liquid-liquid extraction, supported liquid extraction, and solid phase extraction based on both “bind and elute” and “interferent removal” modes, were optimized with an emphasis on the matrix effects and recovery of 8 forms of vitamin E, including α-, β-, γ-, and δ-tocopherols and tocotrienols, from plasma. The matrix effect evaluation included the use and comparison of external and internal calibration using three models, i.e., least square with no transformation and no weighting (1/x0), with 1/x2 weighting, and with logarithmic transformation. The calibration model with logarithmic transformation provided the lowest %-errors and the best fits. Moreover, the type of the calibration model significantly affected not only the fit of the data but also the matrix effects when evaluating them based on the comparison of calibration curve slopes. Indeed, based on the used calibration model, the matrix effects calculated from calibration slopes ranged from +92% to – 72% for α-tocopherol and from −77% to +19% in the case of δ-tocotrienol. Thus, it was crucial to calculate the matrix effect by Matuszewski's post-extraction approach at six concentration levels. Indeed, a strong concentration dependence was observed for all optimized sample preparation methods, even if the stable isotopically labelled internal standards (SIL-IS) were used for compensation. The significant differences between individual concentration levels and compounds were observed, even when the tested calibration range covered only one order of magnitude. In methods with wider calibration ranges, the inappropriate use of calibration slope comparison instead of the post-extraction addition approach could result in false negative results of matrix effects. In the selected example of vitamin E, solid-phase extraction was the least affected by matrix effects when used in interferent removal mode, but supported liquid extraction resulted in the highest recoveries. We showed that the calibration model, the use of a SIL-IS, and the analyte concentration level played a crucial role in the matrix effects. Moreover, the matrix effects can significantly differ for compounds with similar physicochemical properties and close retention times. Thus, in all bioanalytical applications, where different analytes are typically determined in one analytical run, it is necessary to carefully select the data processing in addition to the method for the sample preparation, SIL-IS, and chromatography.

A bivariate fluorescence biosensor based on Janus DNA nanoarchitecture-loaded dual-emissive Ag nanoclusters as bi-responsive signaling reporters

Constructing label-free bivariate fluorescence biosensor would be intriguing and desired for the recognizable and accurate detection of two specific DNA segments, yet the design of functional DNA structures with low overlapped interference might be challenging. Herein in this work, a double-faced Janus DNA nanoarchitecture (JDNA) with bi-responsive recognition regions on opposite sides was assembled, which consisted of two substrate strands and two template strands for loading green-/red-emissive Ag nanoclusters (gAgNC and rAgNC) as bivariate signaling reporters. Of note, the hybridized double helix in the middle rationally oriented two flank faces and stabilized the rigid conformation of JDNA, while the template sequences of bicolor clusters were blocked to minimize non-specific background leakage. Upon inputting two targets, the discernible hairpins lost their hairpin structures due to forming two dsDNA complexes. They were executed to simultaneously invade JDNA for activating two individual target-recycled strand displacement (TRSD) events, guiding signal transduction and efficient amplification. Consequently, the clustering templates were unlocked via the tailored conformation switch of JDNA, in which gAgNC and rAgNC were in situ synthesized in two diagonal positions, thereby significantly emitting bi-responsive signal without cross interference. Benefited from the logic integration of double-faced JDNA and TRSD, a label-free, sensitive and specific bivariate fluorescence approach was developed, which would open a new avenue for the potential application in biosensing and bioanalysis.

Design of a Multiparametric Biosensing Platform and Its Validation in a Study on Spontaneous Cell Detachment from Temperature Gradients.

This article reports on a bioanalytical sensor device that hosts three different transducer principles: impedance spectroscopy, quartz-crystal microbalance with dissipation monitoring, and the thermal-current-based heat-transfer method. These principles utilize a single chip, allowing one to perform either microbalance and heat transfer measurements in parallel or heat transfer and impedance measurements. When taking specific precautions, the three measurement modalities can even be used truly simultaneously. The probed parameters are distinctly different, so that one may speak about multiparametric or "orthogonal" sensing without crosstalk between the sensing circuits. Hence, this sensor allows one to identify which of these label-free sensing principles performs best for a given bioanalytical application in terms of a high signal amplitude and signal-to-noise ratio. As a proof-of-concept, the three-parameter sensor was validated by studying the spontaneous, collective detachment of eukaryotic cells in the presence of a temperature gradient between the QCM chip and the supernatant liquid. In addition to heat transfer, detachment can also be monitored by the impedance- and QCM-related signals. These features allow for the distinguishing between different yeast strains that differ in their flocculation genes, and the sensor device enables proliferation monitoring of yeast colonies over time.

Innovative Colorimetric NQO1 Detection Strategy via Substrate Competitive and Biomimetic Cascade Reactions with a Highly Active NADH Oxidase Mimic.

NAD(P)H: quinone oxidoreductase-1 (NQO1) plays critical roles in antioxidation and abnormally overexpresses in tumors. Developing a fast and sensitive method of monitoring NQO1 will greatly promote cancer diagnosis in clinical practice. This study introduces a transformative colorimetric detection strategy for NQO1, harnessing an innovative competitive substrate mechanism between NQO1 and a new NADH oxidase (NOX) mimic, cobalt-nitrogen-doped carbon nanozyme (CoNC). This method ingeniously exploits the differential consumption of NADH in the presence of NQO1 to modulate the generation of H2O2 from CoNC catalysis, which is then quantified through a secondary, peroxidase-mimetic cascade reaction involving Prussian blue (PB) nanoparticles. This dual-stage reaction framework not only enhances the sensitivity of NQO1 detection, achieving a limit of detection as low as 0.67 μg mL-1, but also enables the differentiation between cancerous and noncancerous cells by their enzymatic activity profiles. Moreover, CoNC exhibits exceptional catalytic efficiency, with a specific activity reaching 5.2 U mg-1, significantly outperforming existing NOX mimics. Beyond mere detection, CoNC serves a dual role, acting as both a robust mimic of cytochrome c reductase (Cyt c) and a cornerstone for enzymatic regeneration, thereby broadening the scope of its biological applications. This study not only marks a significant step forward in the bioanalytical application of nanozymes but also sets the stage for their expanded use in clinical diagnostics and therapeutic monitoring.

Diffusion-driven height readout analytical device based on gold nanobipyramid-doped supramolecular hydrogel for point-of-care bioanalysis

Point-of-care (POC) diagnostics is crucial for rapid on-site detection in resource-limited settings. However, there are still some challenges in the development of quantitative POC bioassays. Herein, we propose a simple, visual, and instrument-free height readout analytical device injected with target-responsive gold nanobipyramid (AuNBP)-doped guanosine-quartet (G4) supramolecular hydrogel for the quantitative detection of glucose. This sensing platform relies on H2O2 generated by glucose oxidation to induce iodide-mediated etching of AuNBPs, resulting in visualized color-changed hydrogel bar-chart driven by molecular diffusion in a polycarbonate tube. The visual height of the color-changed hydrogel is correlated with the concentration of glucose, and the quantitative determination was achieved simply by using a household ruler or mobile software as a readout. More importantly, this method has been successfully used to detect glucose in human serum samples with high accuracy and reliability. In addition, the AuNBPs are utilized as plasmonic probes to construct two-input logic gates, providing an intelligent strategy to simultaneously detect multiple biomolecules. This height-readout sensing platform demonstrates great promise for applications in POC and intelligent bioanalysis.

Thiol-polyethylene glycol-folic acid (HS-PEG-FA) induced aggregation of Au@Ag nanoparticles: A SERS and extinction UV–Vis spectroscopy combined study

Plasmonic colloidal nanoparticles (NPs) functionalised with polymers are widely employed in diverse applications, offering advantages demonstrated over non-functionalised NPs such as enhanced colloidal stability or increased biocompatibility. However, functionalisation with polymers does not always increase the stability of the colloidal system. This work explores the intricate relationship between the functionalisation of plasmonic core@shell Au@Ag nanoparticles (NPs) with thiol-polyethylene glycol-folic acid (HS-PEG-FA) polymer chains and the resulting stability and spectral characteristics of Surface-Enhanced Raman Scattering (SERS) nanotags based on these NPs. We demonstrate that varying levels of HS-PEG-FA grafting influence nanotag stability, with a low level of grafting causing aggregation and subsequently affecting the spectral signature of Raman-reporter molecules attached to the surface of the NP. Electrostatic destabilisation is identified as the primary mechanism driving aggregation, impacting the SERS spectrum of Malachite Green isothiocyanate (MGITC) whose spectral shape is different between the aggregated and non-aggregated NPs. The findings provide valuable insights into NPs stability under different conditions, offering essential considerations for the design and optimisation of SERS nanotags in bio-analytical applications, particularly those involving data processing based on spectral shape, such as in multiplex approaches where experimental spectra are decomposed with several reference components.

Exploring the Application of Terahertz Metamaterials Based on Metallic Strip Structures in Detection of Reverse Micelles.

Terahertz spectroscopy has unique advantages in the study of biological molecules in aqueous solutions. However, water has a strong absorption capability in the terahertz region. Reducing the amount of liquid could decrease interference with the terahertz wave, which may, however, affect the measurement accuracy. Therefore, it is particularly important to balance the amount and water content of liquid samples. In this work, a terahertz metamaterial sensor based on metallic strips is designed, fabricated, and used to detect reverse micelles. An aqueous confinement environment in reverse micelles can improve the signal-to-noise ratio of the terahertz response. Due to "water pool" trapped in reverse micelles, the DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) solution and DOPC emulsion can successfully be identified in intensity by terahertz spectroscopy. Combined with the metamaterial sensor, an obvious frequency shift of 30 GHz can be achieved to distinguish the DOPC emulsion (5%) from the DOPC solution. This approach may provide a potential way for improving the sensitivity of detecting trace elements in a buffer solution, thus offering a valuable toolkit toward bioanalytical applications.