Bioanalytical Applications Research Articles

Development of gene-in-plasmid DNA reference materials certified by single-molecule counting.

The mole, the SI unit for measuring the amount of a substance, was redefined as a fixed number of entities. This definition enables straightforward quantification of substances by counting individual entities. Counting proves particularly effective for quantifying large and discrete biological entities such as DNA, proteins, viruses, and cells, which are challenging to quantify via traditional physical or chemical methods. In this study, we detail our approach to develop gene reference materials certified through single-molecule counting, which enables mole-traceable measurements. We quantified three plasmid DNA constructs, each carrying a specific gene of interest, via single-molecule counting. The resulting values were cross-validated via digital PCR and LC‒MS. Sequence impurities in the certified reference materials were quantified via single-molecule real-time sequencing, whereas fragment impurities were quantified via two-color digital PCR analysis. We precisely accounted for various sources of uncertainty, including measurement precision, weighing, homogeneity, and impurities, when estimating the total uncertainty of the reference materials. In conclusion, a practical format for gene-based DNA reference materials, a measurement method to achieve metrological traceability, and methods for quantifying fragments and sequence impurities were developed and implemented in this study. We anticipate that our gene-based DNA reference materials will serve as valuable higher-order standards for the calibration of other methods or reference materials for DNA quantification in a variety of bioanalytical applications.

Organic photoelectrochemical transistor biosensor based on BiVO4-ZnIn2S4 material for efficient and sensitive detection of MCF-7 cells

Recently, organic photoelectrochemical transistor (OPECT) has become a very interesting biological measurement method in photoelectrochemical (PEC) bioanalysis and future bio-related applications. OPECT is expected to be a powerful tool for disease detection and early warning. Circulating tumor cells (CTCs) are generally deemed to be the dominant factor of tumor metastasis, and 90 % of cancer patients die from this metastatic disease. Therefore, there is an imminent need to develop a highly sensitive CTCs detection sensing system to improve the survival rate of cancer patients. Here, we use a DNA tetrahedrons (DNA NTH) with an aptamer at the top to immobilize on the surface of the photoelectric material to capture cells (MCF-7). Specifically, the BiVO4-ZnIn2S4 hybrid was synthesized by a simple hydrothermal method, which can effectively modulated devices with high current gain. Au NPs were directly integrated on the electrode surface to construct an OPECT photoelectric sensing platform. Subsequently, the aptamer which is thiol-functionalized (SH-Apt) was immobilized on the electrode surface. Because of the overexpression of MUC1 protein on the cell membrane, it can specifically capture MCF-7 cells. The introduction of MCF-7 cells resulted in a significant decrease in the current signal. There is a relationship between the change of photocurrent and the logarithm of MCF-7 cell concentration, which is a good linear relationship ranging from 50 to 5 × 105 cell mL−1. The obtained detection limit is 43 cell mL−1. The biosensor has high selectivity and sensitivity, and achieves sensitive detection of MCF-7.

Embedded optofluidic biosensing platform for enzyme-linked immunosorbent assay

With the growing demand for applications in disease diagnostics, bioanalysis, and health monitoring, developing efficient biosensing systems for fast detections and trace analysis of biomarkers is of great significance. In this work, an embedded optofluidic biosensing platform is proposed, which consists of asymmetrical core-offset optical fiber (ACOOF) structure, microfluidic chip and photoelectric detection system. In particular, by introducing the concept of optical fiber bridge, an ACOOF structure is designed to improve the optical coupling efficiency and reduce the limit of detection (LOD) of the developed sensor. On this basis, the human epidermal growth factor (EGF) was detected by constructing optofluidic chip and enzyme-linked immunosorbent assay (ELISA) kit. The LOD was 0.587 pg/mL. Leveraging the developed biosensing platform integrated with our chip, we achieved trace analysis with high specificity, as demonstrated by the measurement of interleukin 1α (IL-1α) with a low LOD of 43.3 fg/mL. Monitoring these two biomarkers is important for early cancer diagnosis. Beyond this, this novel platform has the potential for a range of applications, including disease diagnosis and management, bioanalysis, and health and environmental monitoring, with a focus on alternative biomarker targets.

Robust In Vitro HPLC-DAD Method for Accurate Quantification of N-Carbamylglutamate in Drug Formulations

A comprehensive in vitro testing method has been developed and validated for accurately quantifying N-Carbamylglutamate (Carglumic acid) using a high-performance liquid chromatography instrument with a diode array detector. A method specifically developed for pharmaceutical quality control utilizes a reverse-phase C18 column for precise and reliable measurement of Carglumic acid. The chromatographic parameters were refined for peak resolution, employing a phosphate buffer and acetonitrile as the mobile phase, with detection setup at 205 nm. The method was validated under ICH Q2(R2) requirements. The approach exhibited a significant level of specificity, with no interference from excipients or other possible impurities. Linearity was achieved across the entire concentration range, yielding an R² value of 0.999. Precision (RSD ≤ 2) was demonstrated by intra- and inter-day variability being within acceptable limits. Recovery tests validated accuracy (98 – 102%). Determining the limit of detection (LOD, 0.1 ppm) and the limit of quantitation (LOQ, 0.5 ppm) was conducted. This in vitro test method was valid and effective for the routine quantification of Carglumic acidin pharmaceutical formulations, serving as a crucial tool for ensuring product quality and regulatory compliance. The methods' elevated sensitivity and endurance make it appropriate for bioanalytical applications, such as dissolution testing and stability analysis.

Dextran-Encapsulated Nanoparticles and Super-Nanoparticle Assemblies: Preparation from Quantum Dots, Fluorescent Polymers, and Magnetic Nanoparticles for Application to Cellular Immunolabeling.

Nanoparticles (NPs) continue to be developed as labels for bioanalysis and imaging due to their small size and, in many cases, emergent properties such as photoluminescence (PL) and superparamagnetism. Some applications stand to benefit from amplification of the advantageous properties of a NP, but this amplification is not a simple matter of scaling for size-dependent properties. One promising approach to amplification is, therefore, to assemble many copies of a NP into a larger but still nanoscale and colloidal entity. Here, we use multiple types of hydrophobic nanocrystal to show that amphiphilic dextran is a versatile material for the preparation and surface functionalization of such super-NP assemblies: CdSe/CdS/ZnS quantum dots (QDs), InP/ZnS QDs, and Si QDs; iron oxide magnetic NPs (MNPs); composites of QDs and MNPs; and composites of QDs and MNPs with fluorene-based and phenylenevinylene-based conjugated polymers. The amphiphilic dextran was also useful for the preparation of conjugated polymer NPs (CPNs) without the inclusion of inorganic nanocrystals. The prepared super-NPs and CPNs were characterized, physically and photophysically, at both the ensemble and the single-particle levels. Per colloidal entity, the super-QDs were orders of magnitude brighter than the individual QDs. This enhancement enabled assemblies of nominally more benign InP/ZnS and Si QDs to be competitive alternative materials to CdSe/CdS/ZnS QDs, which are normally much brighter when compared as individual nanocrystals. The dextran functionalization imparted low nonspecific binding and enabled the use of tetrameric antibody complexes (TACs) for simple and selective immunolabeling of cells with all of the prepared super-NP, CPN, and composite materials. Labeling with the super-QDs provided significantly enhanced PL signals, the super-MNPs enabled magnetic pull-down of cells, and both capabilities were concurrently available with composite assemblies. Overall, this study demonstrates that the preparatory method and functional benefits of amphiphilic dextran extend to a range of hydrophobic materials and combinations thereof. There is strong potential for assembling a diverse set of property-amplified designer labels that are ready-made for in vitro applications in bioanalysis and imaging.

Acoustofluidic Tweezers Integrated with Droplet Sensing Enable Multifunctional Closed-Loop Droplet Manipulation.

Droplet manipulation technologies with surface acoustic waves attract significant attention for applications in fluid handling and bioanalysis. However, existing technologies face challenges in automation, precision, and functional integration, limiting broader applications. In this work, a highly integrated droplet-sensing acoustofluidic tweezer is developed, incorporating orthogonally arranged slanted finger interdigital transducers and a custom-designed control and detection circuit system. Using a single acoustic device, this tweezer enables switchable acoustic droplet manipulation and detection, providing multifunctional closed-loop manipulation of on-chip microliter-scale droplets. The platform takes advantage of the wideband frequency response characteristics of the transducers, along with an automated droplet detection algorithm, enabling high-precision detection of central positions, edge positions, contact diameters, and the number of droplets. With this feedback, automated closed-loop control of various droplet manipulation functions, including transportation, merging, mixing, splitting, and internal particle enrichment, is achieved for the first time on a single acoustic platform. This significantly enhances the precision, efficiency, and fault tolerance of the manipulation process. This droplet-sensing acoustofluidic tweezer provides an innovative acoustic solution for droplet manipulation technologies in fields such as fluid processing and biosensing, demonstrating significant application potential.

Analyte and probe melting temperature guided method development strategy for hybridization LC-MS/MS quantification of siRNAs

Small interfering RNA (siRNA) is a novel class of double-stranded oligonucleotide therapeutics rapidly growing in drug research and development. Accurate, sensitive, and reliable quantification of siRNA analytes in biological samples is required to study their pharmacokinetics, toxicokinetics, and biodistribution. Hybridization LC-MS/MS can achieve highly sensitive and specific bioanalysis of single-stranded oligonucleotides, e.g., antisense oligonucleotides (ASOs); however, its application for bioanalysis of siRNA or other double-stranded oligonucleotides is limited. The detailed rationale and principles for assay development are still not well understood. In this work, we systematically evaluated key steps and parameters of hybridization LC-MS/MS assays, including probes (five different types compared), hybridization procedure and temperature, elution temperature, and column temperature using patisiran, an approved siRNA drug, as the test siRNA. Based on the evaluation, a practical and efficient melting temperature (Tm) guided strategy was developed for fast and reliable method development of hybridization LC-MS/MS assays for siRNA bioanalysis. The strategy was successfully applied to siRNA-A, a test siRNA, in mouse plasma over the range of 1.00–1000 ng/mL and the resulting method has been used to support multiple mouse studies. This method-development strategy showed great value as a general approach for other siRNAs or double-stranded oligonucleotides.

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.

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.