Enzyme Label Research Articles

Nucleotides Bearing Red Viscosity-Sensitive Dimethoxy-Bodipy Fluorophore for Enzymatic Incorporation and DNA Labeling.

Nucleosides and 2'-deoxyribonucleoside triphosphates (dNTPs) bearing 3,3'-dimethoxy-2,2'-diphenyl-6-(4-hydroxyphenyl)-bodipy fluorophore attached through a propargyl or propargyl-triethylene glycol linker to position 5 of 2'-deoxycytidine were designed and synthesized. They exerted bright red fluorescence and good sensitivity to viscosity changing their lifetime from 1.6 to 4.5 ns. The modifed dNTPs were substrates for DNA polymerases and were used in enzymatic synthesis of labeled DNA through primer extension. The modified DNA probes served as viscosity sensors responding to protein binding by changes of lifetime. The nucleotide with longer linker (dCpegMOBTP) was transported to live cells and incorporated into the genomic DNA, which can be useful for staining of DNA and imaging of DNA synthesis.

Fluorescence-Based Analyses of Poly(ADP-Ribose) Length by Gel Electrophoresis, High-Performance Liquid Chromatography, and Capillary Electrophoresis.

Poly(ADP-ribose) (PAR) is a homopolymer made of two or more adenosine diphosphate ribose (ADP-ribose) units. The polymer is usually conjugated to protein as a posttranslational modification playing key roles in cellular processes, such as DNA repair, RNA metabolism, and biomolecular condensate formation. Emergent data revealed that PAR length is highly regulated and determines the selection of and affinity towards protein binders. Here, we describe several fluorescence-based methods that quantify PAR length distributions. Briefly, we use the bioconjugation technique ELTA (enzymatic labeling of terminal ADP-ribose) to fluorescently label PAR, which can be isolated from in vitro and cellular samples. We describe a novel capillary electrophoresis method to separate and quantify PAR length and compare the profile to gel electrophoresis- and high-performance liquid chromatography-based methods. The capillary electrophoresis method is rapid and automatable, enabling accurate determination of the length profiles from subfemtomole quantities of PAR.

Recent Advances in the Immunoassays Based on Nanozymes.

As a rapid and simple method for the detection of multiple targets, immunoassay has attracted extensive attention due to the merits of high specificity and sensitivity. Notably, enzyme-linked immunosorbent assay (ELISA) is a widely used immunoassay, which can provide high detection sensitivity since the enzyme labels can promote the generation of catalytically amplified readouts. However, the natural enzyme labels usually suffer from low stability, high cost, and difficult storage. Inspired by the advantages of superior and tunable catalytic activities, easy preparation, low cost, and high stability, nanozymes have arisen to replace the natural enzymes in immunoassay; they also possess equivalent sensitivity and selectivity, as well as robustness. Up to now, various kinds of nanozymes, including mimic peroxidase, oxidase, and phosphatase, have been incorporated to construct immunosensors. Herein, the development of immunoassays based on nanozymes with various types of detection signals are highlighted and discussed in detail. Furthermore, the challenges and perspectives of the design of novel nanozymes for widespread applications are discussed.

Mitochondrial PARP1 regulates NAD+-dependent poly ADP-ribosylation of mitochondrial nucleoids

PARPs play fundamental roles in multiple DNA damage recognition and repair pathways. Persistent nuclear PARP activation causes cellular NAD+ depletion and exacerbates cellular aging. However, very little is known about mitochondrial PARP (mtPARP) and poly ADP-ribosylation (PARylation). The existence of mtPARP is controversial, and the biological roles of mtPARP-induced mitochondrial PARylation are unclear. Here, we demonstrate the presence of PARP1 and PARylation in purified mitochondria. The addition of the PARP1 substrate NAD+ to isolated mitochondria induced PARylation, which was suppressed by treatment with the inhibitor olaparib. Mitochondrial PARylation was also evaluated by enzymatic labeling of terminal ADP-ribose (ELTA). To further confirm the presence of mtPARP1, we evaluated mitochondrial nucleoid PARylation by ADP ribose-chromatin affinity purification (ADPr-ChAP) and PARP1 chromatin immunoprecipitation (ChIP). We observed that NAD+ stimulated PARylation and TFAM occupancy on the mtDNA regulatory region D-loop, inducing mtDNA transcription. These findings suggest that PARP1 is integrally involved in mitochondrial PARylation and that NAD+-dependent mtPARP1 activity contributes to mtDNA transcriptional regulation.

Tailored calix[4]arene-gold nanoconjugate as a ultra-sensitive immunosensing nanolabel.

The construction of highly sensitive and specific immunosensing nanolabels have attracted tremendous attention in the development of reliable point-of-care disease diagnostics. However, there are still challenges with traditional immunoassays, such as complicated and time-consuming procedure, the use of enzyme label, non-specificity, and require readers for detection. Therefore, we have designed and developed site-directed antibody-immobilized calix[4]arene-gold nanoconjugate based colorimetric immunosensing nanolabel to offer high sensitivity. The prepared nanolabel enabled oriented binding of the antibodies by providing full accessibility of Fab domain for antigen binding. The improved sensitivity of the developed nanolabel was evaluated using vertical flow immunoassay (VFIA) for detecting C-reactive protein (CRP) with a lower detection limit up to 1 ng/ml. Our developed nanolabel was found to be highly specific, easy, quick, and appropriate for onsite detection. The nanolabel is validated with spiked blood samples which exhibited ~90% recovery having a relative error of ~2%. Furthermore, the nanolabel was also used for screening of human blood real samples which showed relative error of ~0.6%. The developed nanolabel can be utilized as a potential nanolabel for the quantitative detection of various biomolecules in clinical samples.

Immunohistochemistry and Immunofluorescence.

Immunohistochemistry (IHC) is one of the most widely used protein detection techniques. The principle of this technique is based on the binding of a specific antibody to a matching specific antigen in tissue. The bound antigen-antibody complex then is visualized using a range of detection techniques. IHC uses a number of different enzymatic labels, such as peroxidase and alkaline phosphatase, for the detection of the antigens of interest whereas immunofluorescence (IF) uses a fluorescent signal. In this chapter, IHC will be described using the peroxidase label. Both IHC and IF can be used on formalin-fixed paraffin-embedded (FFPE) or appropriately processed fresh tissues. IHC/IF can be multiplexed to detect more than one antigen at a time, or may be sequentially stained to detect multiple targets. These techniques are routinely used in diagnostic pathology laboratories, not just for diagnostic purposes but many biomarkers are used for patient staging, treatment allocation, and prognostication. Immunofluorescence is routinely used for the detection of antibodies and antigens in freshly biopsied tissues, particularly for immune-mediated and vesiculobullous lesions. In this chapter, the principles of IHC are reviewed followed by examples of IHC and IF staining using readily available antibodies. Steps and processes involved in IHC/IF double staining are also described.

Electrochemical immunoassay of antibodies using freshly prepared and aged conjugates of silver nanoparticles

In this work, silver nanoparticles (AgNPs) were conjugated with immunoglobulins G using a simple passive adsorption method and the composition and properties of freshly prepared (Ab@AgNPs) and aged (Ab@AgNP/Ag2SNPs) conjugates were investigated for the first time by transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS) and voltammetry. It was found that the state of the AgNPs in the conjugates changed during storage. In the freshly prepared Ab@AgNPs conjugates, silver is present in the metallic state only, while the aged Ab@AgNP/Ag2SNPs conjugates are characterized by the presence of a metallic core of AgNPs coated with a “corona” of Ag2S. This affects the selection of voltammetric conditions for recording an analytical signal from the silver label in the conjugates. The Ab@AgNPs and Ab@AgNP/Ag2SNPs conjugates were successfully tested in the electrochemical immunoassay of dog immunoglobulins as a model. A solid-phase indirect non-competitive assay was performed using a 96-well ELISA plate. To record intense analytical signals from the silver conjugates by linear scan anodic stripping voltammetry (LSASV) on a carbon composite electrode (CCE), they were preliminarily dissolved in acidic solutions. In the case of the Ab@AgNPs conjugate, a nitric acid solution (1 mol L−1) was used to dissolve silver conjugates, while in the case of Ab@AgNP/Ag2SNPs, a 1:1 mixture of 1 mol L−1 CH3COOH and 1 mol L−1 HNO3 was used. The CH3COOH dissolved the Ag2S “corona”, and HNO3 dissolved the silver core of the Ab@AgNP/Ag2SNPs conjugate.This work opens up the possibility of the prolonged use of silver conjugates in electrochemical immunoassay, which can be used in commercial test systems for immunological studies as an alternative to conjugates labelled with enzyme labels.

(Digital Presentation) Rapid and Oxygen-Insensitive Electrochemical Immunoassay for Detection and Staging of Prostate Cancer

Prostate cancer (PCa) is the most common cancer among men diagnosed worldwide. The conventional non-invasive test for detection and staging of prostate cancer include prostate specific antigen (PSA) test which often can give false positives and is unable to distinguish between aggressive and indolent stages of PCa. This results in doing unnecessary invasive tests like digital rectal examination (DRE), biopsy etc., which only gives further anxiety to patients. Here we describe a rapid and oxygen-insensitive microfluidic immunoassay for on-line capture and precise detection of potential prostate cancer protein biomarkers. Measurement of a panel of biomarkers will help in not only diagnosing the PCa precisely but may also help in staging the cancer.The microfluidic system includes protein capture from serum using a sandwich ELISA arrangement and signal-transducing poly-horseradish peroxidase (poly-HRP) enzyme labels on an 8-electrode screen-printed carbon array. A major challenge in using these screen-printed electrode arrays is the estimation of the electrochemical surface area (ECSA) of electrodes because the current signal is proportional to ECSA and variability in the electrode surface areas generate variability in the corresponding currents. We show that normalization of electrode responses having different surface areas can be achieved using in-situ experimental methods and that the normalization methods can improve the precision of the immunoassay. Another challenge in electrochemical immunoassay systems is the interference of oxygen using the mediator hydroquinone1. A range of osmium polypyridyl complexes were studied to find the best mediator that is not affected by oxygen.Using an optimized mediator and a simple method to estimate carbon electrode surface area, immunoassays can be designed with improved detection characteristics.(1) Dhanapala, L.; Jones, A. L.; Czarnecki, P.; Rusling, J. F. Sub-Zeptomole Detection of Biomarker Proteins Using a Microfluidic Immunoarray with Nanostructured Sensors. Anal. Chem. 2020, 92 (12), 8021–8025. ttps://doi.org/10.1021/acs.analchem.0c01507.

An Electrochemical Immunoassay System for Measuring Circulating Protein Biomarkers of Pediatric Soft Tissue Sarcoma

The combination of simple, inexpensive 3D-printed flow cells with electrochemical biosensors has recently emerged a promising strategy for detecting molecular biomarkers of disease. In this study, we report a multiplex electrochemical immunoassay based on pencil graphite electrode arrays, 3D-printed flow cells and magnetic bead bioconjugates for simultaneous detection of three biomarker proteins (cancer antigen 125 (CA-125), midkine (MK) and osteopontin (OPN)) linked to pediatric soft tissue sarcoma (PSTS). Magnetic bead bioconjugates are functionalized with large amounts of antibody and enzyme labels according to a previously reported strategy,1 pencil graphite electrodes are modified with gold nanoparticles and antibodies for specific capture of bioconjugate-labeled biomarkers, and 3D-printed flow cells to facilitate amperometric detection. Using this immunoassay platform, detection limits for CA-125, MK and OPN that are 100 times lower than those obtained using commercial enzyme-linked immunosorbent assay (ELISA) can be achieved. The combination of low-cost pencil graphite electrode arrays with the convenience of multi-labeled magnetic bead bioconjugates and simplicity of 3D-printed flow-cells results in a promising electrochemical biosensing strategy for biomarker proteins CA-125, MK, and OPN in human serum samples.Reference(1) Malhotra, R.; Patel, V.; Chikkaveeraiah, B. v.; Munge, B. S.; Cheong, S. C.; Zain, R. B.; Abraham, M. T.; Dey, D. K.; Gutkind, J. S.; Rusling, J. F. Ultrasensitive Detection of Cancer Biomarkers in the Clinic by Use of a Nanostructured Microfluidic Array. Analytical Chemistry 2012, 84 (14), 6249–6255. https://doi.org/10.1021/ac301392g.

Silver Nanoparticle Labels for Electrochemical Detection of Bioassays

Electrochemistry plays a major role in immunosensor development because of its higher sensitivity, simplicity, portability, and rapidity compared to more common enzymatic, fluorescent and radioactive labelling methods. Silver nanoparticles (AgNPs) have been identified as potential electrochemical labels for such immunoassays. Characterizing the composition, size, shape, and surface modifications of the AgNP is important to achieve expected analytical performance of the bio assay. Here we are interested in studying the effect of nanoparticle shape towards the electrochemical detection of a metallo-immunoassay using Au-Ag galvanic exchange (GE) reaction followed by anodic stripping voltammetry. We observe that during GE between electrogenerated Au3+ and the Ag labels, a thin shell of Au forms on the surface of the NP and prevent further exchange. This shell is more porous when GE proceeds on silver nanocubes (AgNCs) compared to spherical silver nanoparticles (sAgNPs), and therefore, more exchange occurs when using AgNCs. More interestingly, optimizing the ratio of the two types of AgNP labels, we could able to decrease the LOD of a biological assay designed to detect a heart failure biomarker (NT-proBNP) without compromising the dynamic range compared to using either of the two labels independently. This made it possible to achieve the clinically relevant range for NT-proBNP analysis used by physicians for heart failure risk stratification.

Immune Profiling and Multiplexed Label-Free Detection of 2D MXenes by Mass Cytometry and High-Dimensional Imaging.

There is a critical unmet need to detect and image 2Dmaterials within single cells and tissues while surveying a high degree of information from single cells. Here, a versatile multiplexed label-free single-cell detection strategy is proposed based on single-cell mass cytometry by time-of-flight (CyTOF) and ion-beam imaging by time-of-flight (MIBI-TOF). This strategy, "Label-free sINgle-cell tracKing of 2D matErials by mass cytometry and MIBI-TOF Design" (LINKED), enables nanomaterial detection and simultaneous measurement of multiple cell and tissue features. As a proof of concept, a set of 2D materials, transition metal carbides, nitrides, and carbonitrides (MXenes), is selected to ensure mass detection within the cytometry range while avoiding overlap with more than 70 currently available tags, each able to survey multiple biological parameters. First, their detection and quantification in 15 primary human immune cell subpopulations are demonstrated. Together with the detection, mass cytometry is used to capture several biological aspects of MXenes, such as their biocompatibility and cytokine production after their uptake. Through enzymatic labeling, MXenes' mediation of cell-cell interactions is simultaneously evaluated. In vivo biodistribution experiments using a mixture of MXenes in mice confirm the versatility of the detection strategy and reveal MXene accumulation in the liver, blood, spleen, lungs, and relative immune cell subtypes. Finally, MIBI-TOF is applied to detect MXenes in different organs revealing their spatial distribution. The label-free detection of 2D materials by mass cytometry at the single-cell level, on multiple cell subpopulations and in multiple organs simultaneously, will enable exciting new opportunities in biomedicine.

PUP-IT2 as an alternative strategy for PUP-IT proximity labeling.

PUP-IT is a proximity labeling method based on the prokaryotic enzyme PafA. PafA mediates the ligation of Pup, a small peptide, to the proximal proteins. It is different from other proximity labeling methods, such as BioID and APEX, in that both the enzyme and the labeling tag are proteins, which allows for potential in vivo applications. All proximity labeling involves the genetic fusion of the proximity labeling enzyme with the bait protein. However, PafA is a 55 kDa enzyme which sometimes interferes with the bait function. In this study, we tested an alternative proximity labeling strategy, PUP-IT2, in which only a small 7 kDa protein is fused to the bait protein. We examined the activity of PUP-IT2 in vitro and in cells. We also compared it with the original PUP-IT. Finally, we applied PUP-IT2 coupled mass spectrometry to map protein-protein interactions. Overall, we established a new way to use PUP-IT2 for proximity labeling, and this method may have a broad application.

Rapid and reliable RNA resonance assignment by combining chemical and enzymatic stable isotope labeling

In this work a rapid RNA assignment approach by combining chemical and enzymatic 13C and 15N stable isotope labeling is introduced. We exemplify the assignment strategy for imino N1H1 purine and N3H3 pyrimidine and aromatic C6H6 pyrimidine, C8H8 purine and C2H2 adenine resonances for a non-coding RNA comprising 66 nucleotides. The assignment strategy is based on position specific labeling by chemical solid phase synthesis and dilute stable isotope 13C/15N-labeling by mixing labeled and commercially available unlabeled RNA phosphoramidites. The assignment process is further facilitated by nucleotide specific labeling using T7 RNA polymerase in vitro transcription with in house produced atom specific 13C labeled ribonucleotide triphosphates. The approach is fast with a total NMR measurement time of only 22 h and also competitive in terms of costs as compared to the standard methodology relying on in vitro transcription using 2H, 15N and 13C/15N uniformly labeled ribonucleotide triphosphates. Furthermore, the assignment procedure revealed a slow exchange process on the NMR chemical shift time scale in the 66 nt non-coding RNA with possible biological implications in the regulation of bacterial competence.

Development of signal multiplication system for quinone linked immunosorbent assay (Multi-QuLISA) by using poly-l-lysine dendrigraft and 1,2-naphthoquinone-4-sulfonate as enzyme-free tag

A sensitive and stable signal multiplied quinone-linked immunosorbent assay (Multi-QuLISA) was developed. In Multi-QuLISA, an oligomerized quinone linked to biotin, namely biotin-8mer-naphthoquinone (Bio8mer-NQ), is used as a signal-generating label. Bio8mer-NQ is formed from a dendrigraft poly-l-lysine generation 1 (DPLL G1), a controlled branched oligomer composed of eight lysine moieties with nine free amino groups as a backbone. One of the nine amino groups of DPLL G1 is attached to biotin moiety, while the other eight are attached to 1,2-naphthoquinone-4-sulfonate (NQS). Bio8mer-NQ labels a biotinylated detection antibody using avidin as a co-binder. Then, multi-quinones in Bio8mer-NQ undergo a redox cycle with dithiothreitol and luminol, generating strong chemiluminescence. Standard ELISA uses a label enzyme that suffers from vulnerability in different conditions and poor stability. Bio8mer-NQ showed better stability than the enzyme (biotin-HRP) under different drastic pH and temperature conditions, hydrolytic enzymes, etc. Furthermore, Bio8mer-NQ was used as both chemiluminescence and colorimetric label based on the redox cycle of quinone, and it had LODs of 1.5 and 6.5 nM, respectively. The method could detect biotinylated immunocomplex in an in-house designed immunoassay down to 0.2 nM, which is about 25 times more sensitive than biotin HRP. Eventually, Bio8mer-NQ was applied successfully in Multi-QuLISA for detecting β-casein with a sensitivity of 3.2 ng/mL, while the conventional ELISA had an LOD of 35 ng/mL. Overall, Bio8mer-NQ is a stable compound that could be used as an excellent replacement for the enzyme in immunoassay and can be used in both colorimetric and chemiluminescence assays with good sensitivity.

Establishment of an Improved ELONA Method for Detecting Fumonisin B1 Based on Aptamers and Hemin-CDs Conjugates

Fumonisin B1 (FB1) is a strong mycotoxin that is ubiquitous in agricultural products. The establishment of rapid detection methods is an important means to prevent and control FB1 contamination. In this study, an improved enzyme-linked oligonucleotide assay (ELONA) method was designed and tested to detect the contents of FB1 in maize (corn) samples. F10 modified with biotin was bound to an enzyme label plate that was coated with streptavidin (SA) in advance, and carbon dots (CDs) were used to catalyze the color of tetramethylbenzidine (TMB). The complementary chain of F10 was modified with an amino group and coupled with CDs to obtain conjugates. The sample and conjugates were then added to the enzyme plate coated with F10 (an FB1 aptamer). Upon completion of the color reaction, the absorbance was measured at 450 nm. The LOD of this method was 4.30 ng/mL and the LOQ was 13.03 ng/mL. We observed a linear relationship in the FB1 concentration range of 0–100 ng/mL. The standard curve was y = −0.001482 × x + 0.3463, R2 = 0.9918, and the experimental results could be directly measured visually. The recovery of the maize sample was 97.5–99.23% and 94.54–99.25%, and the total detection time was 1 h.

Magnetic relaxation switching immunoassay for chlorpyrifos using enzyme-mediated Fe2+/Fe3+ conversion and magnetic separation

Since pesticide residues in food have attracted increasing concern worldwide, it is crucial to develop rapid and sensitive analytical methods to detect pesticide residues and ensure food safety. In this work, via the biotin-streptavidin (SA) recognition system, we constructed a magnetic relaxation switching (MRS) immunosensor for sensitive detection of chlorpyrifos (CPF). With a competitive immunoassay mode, H2O2 and Fe2+ were firstly optimized as the reaction substrates. Wherein, horseradish peroxidase (HRP) acted as enzyme label to catalyze H2O2 and mediated the conversion of Fe2+/Fe3+. By introducing Fe2+/Fe3+ interconversion as MRS signal output, CPF was detected by the immunoreaction induced variations of the transverse relaxation time (ΔT2). The proposed MRS immunosensor exhibited a detection linear range from 0.01 to 1000 ng mL−1 (R2 = 0.9916), with the limit of detection (LOD) of 6 pg mL−1 (3S/M, n = 3). As a proof-of-concept, CPF was easily detected among five different pesticides at a low concentration level (0.1 ng mL−1), as well as in real samples (apple, tea, and lettuce) with recoveries of 80.70–115.30%. Besides, the sensor can realize one step of “separation and detection” towards CPF with the aid of magnetic nanoparticles, which demonstrate its promising potential for pesticide residue detection in food samples and environment.

Artificial clickase-triggered fluorescence “turn on” based on a click bio-conjugation strategy for the immunoassay of food allergenic protein

Herein, based on an artificial clickase-catalyzed bio-conjugation strategy, we established a sensitive fluorescent clickase-linked immunosorbent assay (FCLISA) platform using an oligonucleotide-molecular beacon (Oligo-MB) hairpin structure as a fluorescence switch for detection of food allergenic protein. Firstly, a highly stable Cu(I)-containing nanocube was prepared for usage as an artificial clickase, which could catalyze the bio-conjugation of two short oligonucleotides (i.e., Oligo-A and Oligo-B labeled by a 5′-alkyne and a 3′-azide group, respectively) through clickase-catalyzed azide/alkyne cycloaddition reaction. Subsequently, the formed long-chain oligonucleotide (Oligo-A-B) could hybridize with Oligo-MB hairpin to open hairpin structure, leading to its fluorescence turn on. By using clickase as an alternative enzymatic label in conventional ELISAs, the established FCLISA showed high sensitivity and accuracy in detection of casein, achieving a limit of detection as low as 1.5 × 10−8 g/mL. Additionally, FCLISA has been challenged by detecting the casein in real samples, indicating a great potential in food safety assay.

A polarized supercell produces specialized metabolites in cannabis trichomes

For centuries, humans have cultivated cannabis for the pharmacological properties that result from consuming its specialized metabolites, primarily cannabinoids and terpenoids. Today, cannabis is a multi-billion-dollar industry whose existence rests on the biological activity of tiny cell clusters, called glandular trichomes, found mainly on flowers. Cannabinoids are toxic to cannabis cells,1 and how the trichome cells can produce and secrete massive quantities of lipophilic metabolites is not known.1 To address this gap in knowledge, we investigated cannabis glandular trichomes using ultra-rapid cryofixation, quantitative electron microscopy, and immuno-gold labeling of cannabinoid pathway enzymes. We demonstrate that the metabolically active cells in cannabis form a "supercell," with extensive cytoplasmic bridges across the cell walls and a polar distribution of organelles adjacent to the apical surface where metabolites are secreted. The predicted metabolic role of the non-photosynthetic plastids is supported by unusual membrane arrays in the plastids and the localization of the start of the cannabinoid/terpene pathway in the stroma of the plastids. Abundant membrane contact sites connected plastid paracrystalline cores with the plastid envelope, plastid with endoplasmic reticulum (ER), and ER with plasma membrane. The final step of cannabinoid biosynthesis, catalyzed by tetrahydrocannabinolic acid synthase (THCAS), was localized in the cell-surface wall facing the extracellular storage cavity. We propose a new model of how the cannabis cells can support abundant metabolite production, with emphasis on the key role of membrane contact sites and extracellular THCA biosynthesis. This new model can inform synthetic biology approaches for cannabinoid production in yeast or cell cultures.

Plasmonic sensing of alkaline phosphatase and its inhibitors through Ag(I)-mediated assembly of gold nanoparticles

Alkaline phosphatase (ALP) is of extraordinary importance in living organisms and widely serves as a labeling enzyme in biochemical assays. Conventional methods based on the colorimetric readout of chemicals have issues in the photosensitivity and the difficulty in visibly distinguishing the color intensity. Herein, we developed a plasmonic assay for detecting ALP activity and its inhibitors based on gold nanoparticles (AuNPs) with good stability and naked-eye readout. Ag(I) specifically oxidizes TMB to produce positively charged oxTMB, which electrostatically aggregates negatively charged AuNPs. ALP as an enzyme specifically hydrolyzes a substrate to generate ascorbic acid, which reduces Ag(I) and modulates the aggregation of AuNPs. This plasmonic ALP assay has a linear detection range of 5–50 mU/mL with a limit of detection of 0.39 mU/mL. In combination with a smartphone, it enables monitoring ALP inhibitors including dimethoate and arsenic that are environmental contaminants. This plasmonic sensing strategy with good stability, visible readout, high specificity, low cost, and ease of operation provides a promising platform for enzyme detection and environmental monitoring.

Fluorescent Platforms for RNA Chemical Biology Research.

Efficient detection and observation of dynamic RNA changes remain a tremendous challenge. However, the continuous development of fluorescence applications in recent years enhances the efficacy of RNA imaging. Here we summarize some of these developments from different aspects. For example, single-molecule fluorescence in situ hybridization (smFISH) can detect low abundance RNA at the subcellular level. A relatively new aptamer, Mango, is widely applied to label and track RNA activities in living cells. Molecular beacons (MBs) are valid for quantifying both endogenous and exogenous mRNA and microRNA (miRNA). Covalent binding enzyme labeling fluorescent group with RNA of interest (ROI) partially overcomes the RNA length limitation associated with oligonucleotide synthesis. Forced intercalation (FIT) probes are resistant to nuclease degradation upon binding to target RNA and are used to visualize mRNA and messenger ribonucleoprotein (mRNP) activities. We also summarize the importance of some fluorescence spectroscopic techniques in exploring the function and movement of RNA. Single-molecule fluorescence resonance energy transfer (smFRET) has been employed to investigate the dynamic changes of biomolecules by covalently linking biotin to RNA, and a focus on dye selection increases FRET efficiency. Furthermore, the applications of fluorescence assays in drug discovery and drug delivery have been discussed. Fluorescence imaging can also combine with RNA nanotechnology to target tumors. The invention of novel antibacterial drugs targeting non-coding RNAs (ncRNAs) is also possible with steady-state fluorescence-monitored ligand-binding assay and the T-box riboswitch fluorescence anisotropy assay. More recently, COVID-19 tests using fluorescent clustered regularly interspaced short palindromic repeat (CRISPR) technology have been demonstrated to be efficient and clinically useful. In summary, fluorescence assays have significant applications in both fundamental and clinical research and will facilitate the process of RNA-targeted new drug discovery, therefore deserving further development and updating.