Bioanalytical Applications Research Articles

Single-Nanoparticle Collision Electrochemistry Biosensor Based on an Electrocatalytic Strategy for Highly Sensitive and Specific Detection of H7N9 Avian Influenza Virus.

Single-nanoparticle collision electrochemistry (SNCE) has gradually become an attractive analytical method due to its advantages in analytical detection, such as a fast response, low cost, low sample consumption, and in situ real-time detection of analytes. However, the biological analyte's direct detection based on the SNCE blocking mode has the problems of low sensitivity and specificity. In this work, an SNCE biosensor based on SNCE electrocatalytic strategy was used for the detection of H7N9 AIV. Nucleic acid aptamers were introduced to recognize the target virus (H7N9 AIV). After the recognition event, ssDNA1 was released and hybridized with another ssDNA2. Owing to the nicking endonuclease Nt.AlwI-mediated target nucleic acid cyclic amplification, one virus particle can indirectly induce the release of 4.2 × 106 Au NPs that can be counted by the SNCE electrocatalytic strategy. The high conversion efficiency greatly improved the detection sensitivity, and the detection limit was as low as 24.3 fg/mL. Therefore, the constructed biosensor can achieve a highly sensitive and specific detection of H7N9 AIV and show a great potential in bioanalytical application.

Sensitive detection of microRNAs using polyadenine-mediated fluorescent spherical nucleic acids and a microfluidic electrokinetic signal amplification chip

The identification of tumor-related microRNAs (miRNAs) exhibits excellent promise for the early diagnosis of cancer and other bioanalytical applications. Therefore, we developed a sensitive and efficient biosensor using polyadenine (polyA)-mediated fluorescent spherical nucleic acid (FSNA) for miRNA analysis based on strand displacement reactions on gold nanoparticle (AuNP) surfaces and electrokinetic signal amplification (ESA) on a microfluidic chip. In this FSNA, polyA-DNA biosensor was anchored on AuNP surfaces via intrinsic affinity between adenine and Au. The upright conformational polyA-DNA recognition block hybridized with 6-carboxyfluorescein-labeled reporter-DNA, resulting in fluorescence quenching of FSNA probes induced by AuNP-based resonance energy transfer. Reporter DNA was replaced in the presence of target miRNA, leading to the recovery of reporter-DNA fluorescence. Subsequently, reporter-DNAs were accumulated and detected in the front of with Nafion membrane in the microchannel by ESA. Our method showed high selectivity and sensitivity with a limit of detection of 1.3 pM. This method could also be used to detect miRNA-21 in human serum and urine samples, with recoveries of 104.0%–113.3% and 104.9%–108.0%, respectively. Furthermore, we constructed a chip with three parallel channels for the simultaneous detection of multiple tumor-related miRNAs (miRNA-21, miRNA-141, and miRNA-375), which increased the detection efficiency. Our universal method can be applied to other DNA/RNA analyses by altering recognition sequences.

One-step synthesis of well-defined molecularly imprinted nanospheres for the class-selective recognition and separation of β-blockers in human serum

β-blockers are a class of medications that are used to treat abnormal heart rhythms and hypertension. Molecularly imprinted polymers (MIPs) capable of selective recognizing and extracting β-blockers from complex biological samples hold great promise in bioanalytical and biomedical applications, but developing such artificial receptor materials is still challenging. Herein, we introduce a simple one-step method for the synthesis of well-defined molecularly imprinted nanospheres in high yield (83.6–94.4%) via reversible addition-fragmentation chain transfer (RAFT) precipitation polymerization for the selective recognition and extraction of the β-blockers from human serum. The prepared MIPs are characterized in terms of morphology, pore properties, binding kinetics, capacity, selectivity, and recognition mechanisms. The uniform nanoscale-imprinted layer favored the rapid mass transfer of β-blockers. The binding studies showed the high adsorption capacity (126.8 μmol/g) and selectivity of the developed nanomaterial. The investigation on the recognition mechanism reveals that multiple driving forces participate in the binding between MIP and β-blockers, where hydrogen bonding plays as the dominating role for the specific recognition. The MIP was successfully applied for the direct enrichment of five β-blockers from human serum with HPLC recoveries ranging from 82.9 to 100.3% and RSD of 0.5–6.9% (n = 3).

Rapid fabrication of zwitterionic sulfobetaine vinylimidazole-based monoliths via photoinitiated copolymerization for hydrophilic interaction chromatography

Zwitterionic sulfobetaine-based monolithic stationary phases have attracted increasing attention for their use in hydrophilic interaction chromatography. In this study, a novel hydrophilic polymeric monolith was fabricated through photo-initiated copolymerization of 3-(3-vinyl-1-imidazolio)-1-propanesulfonate (SBVI) with pentaerythritol triacrylate using methanol and tetrahydrofuran as the porogenic system. Notably, the duration for the preparation of this novel monolith was as little as 5 min, which was significantly shorter than that required for previously reported sulfobetaine-based monoliths prepared via conventional thermally initiated copolymerization. Moreover, these monoliths showed good morphology, permeability, porosity (62.4%), mechanical strength (over 15 MPa), column efficiency (51,230 plates/m), and reproducibility (relative standard deviations for all analytes were lower than 4.6%). Mechanistic studies indicated that strong hydrophilic and negative electrostatic interactions might be responsible for the retention of polar analytes on the zwitterionic SBVI-based monolith. In particular, the resulting monolith exhibited good anti-protein adhesion ability and low nonspecific protein adsorption. These excellent features seem to favor its application in bioanalysis. Therefore, the novel zwitterionic sulfobetaine-based monolith was successfully employed for the highly selective separation of small bioactive compounds and the efficient enrichment of N-glycopeptides from complex samples. In this study, we prepared a novel zwitterionic sulfobetaine-based monolith with good performance and developed a simpler and faster method for preparation of zwitterionic monoliths.

3D-printed electrochemical platform with multi-purpose carbon black sensing electrodes.

The 3D printing is described of a complete and portable system comprising a batch injection analysis (BIA) cell and an electrochemical platform with eight sensing electrodes. Both BIA and electrochemical cells were printed within 3.4 h using a multimaterial printer equipped with insulating, flexible, and conductive filaments at cost of ca. ~ U$ 1.2 per unit, and their integration was based on a threadable assembling without commercial component requirements. Printed electrodes were exposed to electrochemical/Fenton pre-treatments to improve the sensitivity. Scanning electron microscopy and electrochemical impedance spectroscopy measurements upon printed materials revealed high-fidelity 3D features (90 to 98%) and fast heterogeneous rate constants ((1.5 ± 0.1) × 10−3 cm s−1). Operational parameters of BIA cell were optimized using a redox probe composed of [Fe(CN)6]4−/3− under stirring and the best analytical performance was achieved using a dispensing rate of 9.0 µL s−1 and an injection volume of 2.0 µL. The proof of concept of the printed device for bioanalytical applications was evaluated using adrenaline (ADR) as target analyte and its redox activities were carefully evaluated through different voltammetric techniques upon multiple 3D-printed electrodes. The coupling of BIA system with amperometric detection ensured fast responses with well-defined peak width related to the oxidation of ADR applying a potential of 0.4 V vs Ag. The fully 3D-printed system provided suitable analytical performance in terms of repeatability and reproducibility (RSD ≤ 6%), linear concentration range (5 to 40 µmol L−1; R2 = 0.99), limit of detection (0.61 µmol L−1), and high analytical frequency (494 ± 13 h−1). Lastly, artificial urine samples were spiked with ADR solutions at three different concentration levels and the obtained recovery values ranged from 87 to 118%, thus demonstrating potentiality for biological fluid analysis. Based on the analytical performance, the complete device fully printed through additive manufacturing technology emerges as powerful, inexpensive, and portable tool for electroanalytical applications involving biologically relevant compounds.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00604-022-05323-4.

An Organoplatinum(II) Metallacycle-Based Supramolecular Amphiphile and Its Application in Enzyme-Responsive Controlled Release.

Enzyme-responsive nanomaterials are emerging as important candidates for bioanalytical and biomedical applications due to their good biocompatibilities and sensitivities. However, the lack of promising operation platforms compatible with enzyme responsiveness greatly limits the scope and functionality of smart materials. Herein, we report the design and synthesis of a naphthalene-functionalized organoplatinum(II) metallacycle 1 by means of coordination-driven self-assembly, which is subsequently exploited as the organometallic platform to enable enzyme-responsive supramolecular materials. Specifically, a [2 + 2] self-assembled metallacycle 1 first self-assembles into nanosheets in aqueous solution, which can further transform into vesicles with the introduction of β-cyclodextrin (β-CD) because of the formation of a bola-type supramolecular amphiphile β-CD-1. Interestingly, these vesicles show rare α-amylase responsiveness, as demonstrated by structurally transforming back into nanosheets after the addition of α-amylase to their solutions due to the enzyme-induced degradation of cyclodextrins. We also demonstrate the potential application of the self-assembled vesicles in amylase-responsive controlled release.

DNA Templated Silver Nanoclusters for Bioanalytical Applications: A Review

Due to their unique programmability, biocompatibility, photostability and high fluorescent quantum yield, DNA templated silver nanoclusters (DNA Ag NCs) have attracted increasing attention for bioanalytical application. This review summarizes the recent developments in fluorescence properties of DNA templated Ag NCs, as well as their applications in bioanalysis. Finally, we herein discuss some current challenges in bioanalytical applications, to promote developments of DNA Ag NCs in biochemical analysis.

Extension of duplex specific nuclease sensing application with RNA aptamer.

Duplex specific nuclease (DSN) that can precisely cleave DNA portion in double-stranded DNA or DNA-RNA hybrid has engrossed immense attention owing to its great potential in emerging bioanalytical applications. Here, we present a novel approach to extend DSN sensing application by coupling RNA aptamer. Specially designed RNA ligand sequences are used to capture the target and simultaneously provide complementary sequences of DNA for DSN aided fluorescent signal enhancement. A clotting enzyme, thrombin, has been used as a model analyte. One RNA aptamer combined with the target molecule can generate fluorescent signals through cleavage of hybridized TaqMan DNA probe (P2) by DSN. The proposed assay has achieved the lowest detection limit of 0.039 pM. The assay has been applied for real-time detection of thrombin release from live cells and other biotic media for early disease diagnosis. The developed method is versatile and can detect various other targets by choosing the relevant aptamer and probe sequences. This method is promising to be applied to medical diagnosis, biosensing, food safety, environmental monitoring, and other fields.

Numerical Modelling of Mixing in a Microfluidic Droplet Using a Two-Phase Moving Frame of Reference Approach.

Droplets generated in microfluidic channels are effective self-contained micromixers and micro-reactors for use in a multiplicity of chemical synthesis and bioanalytical applications. Droplet microfluidic systems have the ability to generate multitudes of droplets with well-defined reagent volumes and narrow size distributions, providing a means for the replication of mixing within each droplet and thus the scaling of processes. Numerical modelling using computational fluid dynamics (CFD) is a useful technique for analysing and understanding the internal mixing in microfluidic droplets. We present and demonstrate a CFD method for modelling and simulating mixing between two species within a droplet travelling in straight microchannel, using a two-phase moving frame of reference approach. Finite element and level set methods were utilised to solve the equations governing the coupled physics between two-phase flow and mass transport of the chemical species. This approach had not been previously demonstrated for the problem of mixing in droplet microfluidics and requires less computational resources compared to the conventional fixed frame of reference approach. The key conclusions of this work are: (1) a limitation of this method exists for flow conditions where the droplet mobility approaches unity, due to the moving wall boundary condition, which results in an untenable solution under those conditions; (2) the efficiency of the mixing declines as the length of the droplet or plug increases; (3) the initial orientation of the droplet influences the mixing and the transverse orientation provides better mixing performance than the axial orientation and; (4) the recirculation inside the droplet depends on the superficial velocity and the viscosity ratio.

Recent Advances on Nanozyme‐based Electrochemical Biosensors

AbstractNanozymes are nanomaterials with enzyme‐like catalytic activities. The unique features of nanozymes (such as high stability, low cost, large surface area for bioconjugation, ease of storage, and multi‐functionalities) offer unprecedented opportunities for designing electrochemical biosensors. Recent years have witnessed the rapid development of nanozyme‐based electrochemical biosensors. To highlight these achievements, this review first discusses the representative nanozymes including peroxidase mimics, oxidase mimics, hydrolase mimics, and superoxide dismutase mimics used in electrochemical biosensors. Then, it summarizes the bioanalytical applications for the detection of various analytes. Finally, current challenges and future research directions are summarized.

Construction of a bioinspired Fe3O4/N-HCS nanozyme for highly sensitive detection of GSH

The catalytic synergy of different nanomaterials is a potential technique used to develop a highly active and stable nanozyme. Here, magnetite on nitrogen-doped hollow carbon sphere (Fe3O4/N-HCS) composites were prepared through solvent thermal method and exhibited excellent oxidase and peroxidase-like catalytic activity under different pH levels. The dual enzyme-like catalyzed reaction of Fe3O4/N-HCS conformed to the typical Michaelis–Menten kinetics, and Fe3O4/N-HCS had a strong affinity for substrate 3,3′,5,5′-tetramethylbenzidine (TMB) with H2O2. The remarkable performance of the composites was achieved by the synergistic effect of Fe3O4 and N-doped activated graphite carbon, which accelerated the formation of O2•- and •OH during the dual enzyme-like catalytic reaction. Furthermore, an effective colorimetric glutathione (GSH) sensor with a wide linear range from 0.02 µM to 250 µM and a detection limit as low as 9.3 nM was constructed based on the superior peroxidase-like activity of Fe3O4/N-HCS. The convenient colorimetric sensor monitored the content of GSH in the serum of mice, and the detection results were satisfactory, with a recovery of 96–103% and an RSD between 0.80% and 3.30%. This study discussed the internal mechanism of Fe3O4/N-HCS nanozyme and developed an efficient method for GSH detection to expand nanozyme application in bioanalysis.

Synthesis of β-cyclodextrin grafted rhombohedral-CuO antioxidant nanozyme for detection of dopamine and hexavalent chromium through off–on strategy of peroxidase mimicking activity

For the last few decades, peroxidase mimicking inorganic nanoparticles are widely used as compared to natural peroxidase due to some basic advantages especially high durability, stability in harsh conditions, easy and cost-effective synthesis, and easy storage. However, most of these peroxidase mimicking nanoparticles are not always effective in a neutral medium, most of the show high activity in an acidic medium which seriously impedes their uses in bioanalytical applications. In the current study, we have developed an inorganic β-cyclodextrin (β-CD) grafted rhombohedral cupric oxide (R-CuO) nanozyme which showed high catalytic activity at neutral conditions (pH = 7) and also exhibited catalytic activity in a wide range of pH with enzymatic substrate TMB (3,3–5,5 tetramethylbenzidine).The current nanozyme has exhibited outstanding peroxidase mimic activity with low Km and very high Vmax value which was calculated by the double reciprocal Lineweaver Burk plot. Considering the R-CuO NPs as a heterogeneous catalyst, a one-pot simple colorimetric system has been developed for dopamine detection, as dopamine turns off the catalytic activity of the R-CuO NPs-TMB peroxidase system. The sensitivity of this current probe to dopamine was promising with a linear range of 2– 24 µM and had a low detection limit of 1.91 µM. More surprisingly, the R-CuO-TMB /dopamine system undergoes recovery of peroxidase activity upon introduction of Cr (+6) (dichromate).The method displays a good selectivity towards Cr (+6) (dichromate) over all the other metal ions with a wide linear relationship in the range of 2 μM–10 μM, and a low detection limit of 0.47 μM. Moreover, the present Nano system was not only applicable for toxic metal and bioactive molecules detection they also used as a ROS scavenger. Our Nano system exhibited scavenging activity for OH radical, O2–. species and metal chelation activity which was investigated calorimetrically. So, the present Nano system is used as a colorimetric probe for dopamine and Cr (+6) (dichromate) detection and consumption of reactive oxygen species as well.

Synthesis of KCl-doped lignin carbon dots nanoenzymes for colorimetric sensing glutathione in human serum

A KCl-doped lignin carbon dots (K,Cl-LCDs) nanoenzymes was successfully synthesized by hydrothermal method to construct a sensor for detecting glutathione (GSH). Sodium lignosulfonate with a lot of oxygen-containing functional groups as a carbon precursor can provide effective active sites, and KCl as a dopant can influence the charge distribution on the surface of CDs. In the enzyme catalyzing progress, K,Cl-LCDs with peroxidase-like activity can oxidize 3,3,5,5-tetramethylbenzidine (TMB) into blue oxTMB with the presence of H2O2, the absorbance of the system at 652 nm changed consequently. When GSH is introduced into the system, the reducibility of GSH restores oxTMB to colorless. So that, a sensor for colorimetric detection of GSH is constructed. In addition, through free radical experiments, the catalytic mechanism was explored, confirming that the reaction was induced by hydroxyl radicals (·OH). The sensor has a low detection limit (0.08 μM) for detecting GSH with good linear range (0.25–25 μM). The sensor system was applied to the determination of GSH concentration in human serum samples with the recoveries of 98.2%− 103.2% and RSDs of 2.3%− 4.7%. The proposed sensor for GSH detection has the advantages of simple operation, low cost, and high speed, indicating its potential application in bioanalysis.

About this issue

Modern analytical chemistry comprises an amazing scope of techniques and methods used for the characterization of analytes in complex matrices. Despite the numerous formats and instrument configurations, the challenges faced by analysts in innovation-driven environments are, in essence, the same. Recent advances in separation science have imposed new requirements for sampling and sample preparation, which has ultimately altered the essence of contemporary sample preparation. Advances in column technology and multidimensional instrumentation has led to the achievement of unprecedented peak capacities. The evolution of hyphenated methods has enabled the generation of information-dense data tensors, which ultimately impact the fundamental role and formats available for data processing and interpretation (see Issue #32). This paradigm shift has created exciting opportunities for analytical chemistry. In this issue #35, we have carefully selected applications covering some of the most important steps of an analytical method, namely, sample preparation and method development using separation and spectroscopy to solve real world tasks. For instance, a review article covers the potential of combining restricted access materials with molecularly imprinted polymers for bioanalytical applications. This article is followed by a report on the extraction and characterization of Tamarind gum from an unusual source of biomass, which is an important step towards the circular economy. Next, applications of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and inductively coupled plasma mass spectrometry (ICP-MS) are also available for the interested reader. In this issue, we have also interviewed Dr. Gisele Tonietto about the dissemination of science, published a point of view with Dr. Emanuel Carrilho, and a letter from Dr. Cesar Tarley. Lastly, I would like to thank all the authors, reviewers, and editorial staff for putting this much effort and dedication into publishing this issue on time. This journal has grown beyond expectations, while accompanying the evolution of research and innovation in Brazil. It is our hope to continue pushing the boundaries of science by inviting interested authors from Latin American and the world. I look forward to publishing manuscripts that will also recognize and promote equality in science, highlighting role models for younger generations of researchers.

Ultrafast synthesized monometallic nanohybrids as an efficient quencher and recognition antenna of upconversion nanoparticles for the detection of xanthine with enhanced sensitivity and selectivity

Upconversion nanoparticles (UCNPs) have shown great promise in bioanalytical applications owing to their excellent optical properties. Generally, most analytical applications are based on the fluorescence resonance energy transfer (FRET) principle to quench the fluorescence of UCNPs. However, each UCNP contains thousands of emission center ions, and most of them exceed the FRET critical distance, which hinders FRET efficiency and leads to a low signal-to-background ratio (SBR). Herein, a novel nanoprobe for the detection of Xanthine (XA) based on inner filter effects (IFE) and cascade signal amplification strategy was constructed by decorating UCNP with trypsin-chymotrypsin-stabilized gold nanoparticles-gold nanoclusters (Try-chy-AuNPs-AuNCs) monometallic nanohybrids. The Try-chy-AuNPs-AuNCs prepared by ultrafast (3 min) and green synthesis method have efficient upconversion fluorescence quenching ability (the quenching efficiency up to 90.9%), which can effectively improve the SBR of the probe, so as to improve the sensitivity. In addition, the Try-chy-AuNPs-AuNCs have a unique spatial structure, which can effectively prevent the interaction between large-size biothiol (glutathione) and the probe, thus improving its selectivity. Besides, combined with the excellent optical performance of UCNPs and cascaded signal amplification strategy, the sensitivity of the probe can be further improved. Under the optimized conditions, the linear response range of the probe was obtained from 0.05 to 50 μM, 0.06–80 μM and with the low detection limit of 22.6 nM and 26.3 nM for H2O2 and XA, respectively. Meanwhile, the developed method has been further applied to the detection of XA in human serum with satisfactory results.

Quantitative analysis of favipiravir and hydroxychloroquine as FDA-approved drugs for treatment of COVID-19 using synchronous spectrofluorimetry: application to pharmaceutical formulations and biological fluids.

Coronavirus disease 2019 (COVID‐19) is a contagious viral infection caused by coronavirus 2 (SARS‐CoV‐2) that causes severe acute respiratory syndrome. It has ravaged several countries and burdened many healthcare systems. As the process of authorizing a novel treatment for human use is extensive and involves multiple phases to obtain safety information and identify potential concerns. Therefore, the fastest and easiest choice was to use United States Food and Drug Administration (US FDA)‐approved drugs such as favipiravir and hydroxychloroquine. For the simultaneous estimation of both medications, a simple synchronous spectrofluorimetric approach was established in which both drugs were measured at 372 and 323 nm, respectively in the presence of each other without interference at Δλ 60 nm. The effect of various experimental conditions on synchronous fluorescence intensities were thoroughly investigated and optimized. The maximum synchronous fluorescence intensities were obtained at pH 5.4 using acetate buffer (0.2 M, 0.5 ml) and ethanol as a diluent. Excellent linearity ranges were obtained using 1.0–18.0 ng/ml and 10.0–120.0 ng/ml for favipiravir and hydroxychloroquine, respectively. The approach exhibited high sensitivity with detection limits down to 0.25 ng/ml and 1.52 ng/ml and quantitation limits down to 0.77 ng/ml and 4.62 ng/ml, respectively. Spiking human plasma samples with the studied drugs yielded high % recoveries, allowing a significant bioanalytical application. Moreover, the method was validated according to International Conference on Harmonization guidelines and further applied to commercial pharmaceutical preparations with good results.

Stripping voltammetry and chemometrics assisted ultra-selective, simultaneous detection of trace amounts of heavy metal ions in aqua and blood serum samples

We present a one-step, solid-state synthesis of Al2NiCoO5 nanoflakes (ANC) modified glassy carbon electrode (GCE) for ultra-selective and trace level simultaneous detection of HMIs like copper (Cu2+), mercury (Hg2+), cadmium (Cd2+), and lead (Pb2+) ions in aqua and simulated blood serum samples. ANC/GCE sensor exhibits a low limit of detection (LOD = 3S/m) of 0.00154 ppb, 0.00232 ppb, 0.00261 ppb and 0.00114 ppb towards Pb2+, Hg2+ Cu2+ and Cd2+ ions, correspondingly that are far lower than the perilous limits of HMI concentration reported for blood serum and water samples. The quantitative study and multiple response analysis of the sensor towards each metal ions are accomplished via principal component analysis (PCA) which verifies the selectivity of the ANC/GCE sensor with satisfactory recovery percentages. The superior performance, reproducibility and stability of the sensor are attributed to the electrocatalytic active sites of ANC nanoflakes facilitated by the Ni2+/Ni3+ and Co2+/Co3+ redox couple along with the oxy-functional groups. The sensor was efficacious in detecting the trace level concentrations of HMIs in samples like simulated blood serum, drinking water and tap water with good recovery percentages. This efficiency of the sensor demonstrates it as a promising platform for a broad-spectrum of environmental and bioanalytical applications.

Ratiometric electrochemical immunosensor triggered by an advanced oxidation process for the ultrasensitive detection of carcinoembryonic antigen

Conventional electrochemical immunosensors often achieve detection purposes through a single signal output, but its electrical signal is susceptible to external environmental interference, which seriously interferes with the sensitivity and repeatability of the sensors. In this work, a novel ratiometric electrochemical immunosensor triggered by an advanced oxidation process (AOP) was developed for the ultrasensitive detection of carcinoembryonic antigen (CEA). The sensor uses gold nanoparticle-reduced graphene oxide/methylene blue adsorbed by sodium alginate (Au-rGO/MB-SA) as matrix to immobilize primary antibody (Ab1) and Ag nanoparticles-functionalized egg yolk structure Co3O4@SiO2 (Co3O4-Ag@SiO2) as a marker to label secondary antibody (Ab2). When the detection solution contains peroxymonosulfate (PMS), the signal probe Co3O4-Ag@SiO2 activates PMS to produce SO4•−/•OH radicals to degrade MB, thus generating two independent electrochemical signals with opposite directional changes (IAg and IMB). The sensitivity of the sensor is improved due to an enhanced response magnitude on account of the processing of the dual selective responsive signals to yield a response magnitude greater than that of the signal from a single sensor. Square wave voltammetry (SWV) analysis shows that the dual signal ratio (I = IAg/IMB) has a linear relationship with the logarithm of the CEA concentration in the range of 0.01 pg/mL to 40 ng/mL, and the detection limit is as low as 3.42 fg/mL. In addition, the sensor has good selectivity and reproducibility, showing great promise for applications in bioanalysis and disease diagnosis.

DIRECT2: A novel platform for a CRISPR–Cas12-based assay comprising universal DNA–IgG probe and a direct lateral flow test

CRISPR-Cas12-based biosensors are a promising tool for the detection of nucleic acids. After dsDNA-target-activated Cas12 cleaves the ssDNA probe, a lateral flow test (LFT) is applied for rapid, simple, and out-of-laboratory detection of the cleaved probe. However, most of the existing approaches of LFT detection have disadvantages related to inverted test/control zones in which the assay result depends not only on the cleavage of the probe but also on the second factor: the binding of the non-cleaved probe in the control zone. We proposed a novel platform for the detection of trans-cleaved DNA using a universal DNA-IgG probe and LFT with the sequential direct location of test and control zones. The advantage of the platform consists of the assay result depending only on the cleaved probe. For this, we designed a composite probe that comprise two parts: the DNA part (biotinylated dsDNA connected to ssDNA with fluorescein) (FAM), and the antibody part (mouse anti-FAM IgG). The Cas12, with guide RNA, was activated by the dsDNA-target. The activated Cas12 cleaved the probe, releasing the ssDNA-FAM-IgG reporter that was detected by the LFT. The sandwich LFT was proposed with anti-mouse IgG adsorbed in the test zone and on the surface of gold nanoparticles. We called the platform with direct location zones and direct analyte-signal dependence the DNA-Immunoglobulin Reporter Endonuclease Cleavage Test (DIRECT2). Therefore, this proof-of-concept study demonstrated that the combination of the proposed DNA-IgG probe and direct LFT opens new opportunities for CRISPR–Cas12 activity detection and its bioanalytical applications.

PMVEMA-coated upconverting nanoparticles for upconversion-linked immunoassay of cardiac troponin

Surface engineering of upconverting nanoparticles (UCNPs) is crucial for their bioanalytical applications. Here, an antibody specific to cardiac troponin I (cTnI), an important biomarker for acute myocardial infection, was covalently immobilized on the surface of UCNPs to prepare a label for the detection of cTnI biomarker in an upconversion-linked immunoassay (ULISA). Core-shell UCNPs (NaYF4:Yb,Tm@NaYF4) were first coated with poly(methyl vinyl ether‐alt‐maleic acid) (PMVEMA) and then conjugated to antibodies. The morphology (size and uniformity), hydrodynamic diameter, chemical composition, and amount of coating on the of UCNPs, as well as their upconversion luminescence, colloidal stability, and leaching of Y3+ ions into the surrounding media, were determined. The developed ULISA allowed reaching a limit of detection (LOD) of 0.13 ng/ml and 0.25 ng/ml of cTnI in plasma and serum, respectively, which represents 12- and 2-fold improvement to conventional enzyme-linked immunosorbent based on the same immunoreagents.