A high-throughput differential scanning fluorimetry method for rapid detection of thermal stability and iron saturation in lactoferrin.

Thermal stability and iron saturation of lactoferrin (LF) are of great significance not only for the evaluation of the biological activities of LF but also for the optimization of the isolation and drying process parameters. Differential scanning calorimetry (DSC) is a well-established and efficient method for thermal stability and iron saturation detection in LF. However, multiple DSC measurements are typically performed sequentially, thus time-consuming and low throughput. Herein, we introduced the differential scanning fluorimetry (DSF) approach to overcome such limitations. The DSF can monitor LF thermal unfolding with a commonly available real-time PCR instrument and a fluorescent dye (SYPRO orange or Glomelt), and the measured melting temperature of LF is consistent with that determined by DSC. On the basis of that, a new quantification method was established for determination of iron saturation levels using the linear correlation of the degree of ion saturation of LF with DSF measurements. Such DSF method is simple, inexpensive, rapid (<15 min), and high throughput (>96 samples per experiment), and provides a valuable alternative tool for thermal stability detection of LF and other whey proteins.

Recent Advances in Gold Nanocluster-Based Biosensing and Therapy: A Review.

Gold nanoclusters (Au NCs) with bright emission and unique chemical reactivity characters have been widely applied for optical sensing and imaging. With a combination of surface modifications, effective therapeutic treatments of tumors are realized. In this review, we summarize the recently adopted biosensing and therapy events based on Au NCs. Homogeneous and fluorometric biosensing systems toward various targets, including ions, small molecules, reactive oxygen species, biomacromolecules, cancer cells, and bacteria, in vitro and in vivo, are presented by turn-off, turn-on, and ratiometric tactics. The therapy applications are concluded in three aspects: photodynamic therapy, photothermal therapy, and as a drug carrier. The basic mechanisms and performances of these systems are introduced. Finally, this review highlights the challenges and future trend of Au NC-based biosensing and therapy systems.

Ultrasensitive NIR fluorometric assay for inorganic pyrophosphatase detection via Cu2+-PPi interaction using bimetallic Au-Ag nanoclusters.

BACKGROUND: Inorganic pyrophosphatase (PPase) is key enzyme playing a key role in biochemical transformations such as biosynthesis of DNA and RNA, bone formation, metabolic pathways associated with lipid, carbohydrate and phosphorous. It has been reported that lung adenocarcinomas, colorectal cancer, and hyperthyroidism disorders can result from abnormal level of PPase. Therefore, it is of notable significance to develop simple and effective real time assay for PPase enzyme activity monitoring for screening of many metabolic pathways as well as for early disease diagnosis. RESULT: The fluorometric detection of PPase enzyme in near infrared region-1 (NIR-1) has been carried out using bimetallic nanoclusters (LA@AuAg NCs). The developed sensing strategy was based on quenching of fluorescence intensity of LA@AuAg NCs upon interaction with copper (Cu2+) ions. The off state of LA@AuAg_Cu2+ ensemble was turned on upon addition of pyrophosphate anion (PPi) due to strong binding interaction between PPi and Cu2+. The catalytic conversion of PPi into phosphate anion (Pi) in the presence of PPase led to liberation of Cu2+ ions, and again quenched off state was retrieved due to interaction of free Cu2+ with LA@AuAg NCs. The ultrasensitive detection of PPase was observed in the linear range of 0.06-250 mU/mL with LOD as 0.0025 mU/mL. The designed scheme showed good selectivity towards PPase enzyme in comparison to other bio-substrates, along with good percentage recovery for PPase detection in real human serum samples. SIGNIFICANCE: The developed NIR based assay is ultrasensitive, highly selective and robust for PPase enzyme and can be safely employed for other enzymes detection. This highly sensitive nature of biosensor was result of involvement of fluorescence-based technique and synergistic effect of dual metal in NIR based bimetallic NCs. Moreover, owing to the emission in NIR domain, in future, these nanoclusters can be safely employed for many biomedical applications for In vivo studies.

ß-Glucuronidase-triggered reaction for fluorometric and colorimetric dual-mode assay based on the in situ formation of silicon nanoparticles.

BACKGROUND: ß-Glucuronidase (GUS) is considered as a promising biomarker for primary cancer. Thus, the reliable detection of GUS has great practical significance in the discovery and diagnosis of cancer. Compared with traditional organic probes, silicon nanoparticles (Si NPs) have emerged as robust optical nanomaterials due to their facile preparation, superior photobleaching resistance and excellent biocompatibility. However, most nanomaterials-based methods only output a single signal which is easily influenced by external factors in complex systems. Hence, developing nanomaterial-based multi-signal optical assays for highly sensitive GUS determination is still urgently desired. RESULTS: In this study, we developed a simple and efficient one-step method for the in situ preparation of yellow color and yellow-green fluorescent Si NPs. This was achieved by combining 3-[2-(2-aminoethylamino) ethylamino] propyl-trimethoxysilane with p-aminophenol (AP) in an aqueous solution. The obtained Si NPs showed yellow-green fluorescence at 535 nm when excited at 380 nm, while also exhibiting an absorption peak at a wavelength of 490 nm. Taking inspiration from the easy synthesis step regulated by AP, which is generated through the hydrolysis of 4-aminophenyl ß-D-glucuronide catalyzed by GUS, we constructed a direct fluorometric and colorimetric dual-mode method to measure GUS activity. The developed fluorometric and colorimetric sensing platform showed high sensitivity and accuracy with detection limits for GUS determination as low as 0.0093 and 0.081 U/L, respectively. SIGNIFICANCE: This study provides a facile dual-mode fluorometric and colorimetric approach for determination of GUS activity based on novel Si NPs for the first time. This designed sensing approach was successfully employed for the quantification of GUS in human serum samples and screening of GUS inhibitors, indicating the feasibility and potential applications in clinical cancer diagnosis and anti-cancer drug discovery.

A sensitive fluorometric-colorimetric dual-mode intelligent sensing platform for the detection of formaldehyde.

Excess formaldehyde (FA) is a strong carcinogen, so the development of a rapid visualized and portable formaldehyde detection platform is of great research importance. A multi-color fluorescence sensing system constituted of model compound (NAHN) and red-emitting InP/ZnS QDs was constructed herein, which can simultaneously realize fluorometric-colorimetric dual-mode sensing when exposed to FA environment. Its preparation process was simplified, the detection process was green, and the limits of detection (LOD) were 0.623 µM and 0.791 µM, respectively. The high recoveries of FA in actual water samples indicated that the sensor had broad application prospects. The prepared fluorescent film can be utilized for rapid visual simulation analysis of FA on the surface of various fruits and vegetables. In addition, a serial logic gate was designed to quickly semi-quantitatively assess FA concentration, which promoted the realization of on-site intelligent evaluation of FA.

Isothermal chemical denaturation assay for monitoring protein stability and inhibitor interactions.

Thermal shift assay (TSA) with altered temperature has been the most widely used method for monitoring protein stability for drug research. However, there is a pressing need for isothermal techniques as alternatives. This urgent demand arises from the limitations of TSA, which can sometimes provide misleading ranking of protein stability and fail to accurately reflect protein stability under physiological conditions. Although differential scanning fluorimetry has significantly improved throughput in comparison to differential scanning calorimetry and differential static light scattering throughput, all these methods exhibit moderate sensitivity. In contrast, current isothermal chemical denaturation (ICD) techniques may not offer the same throughput capabilities as TSA, but it provides more precise information about protein stability and interactions. Unfortunately, ICD also suffers from limited sensitivity, typically in micromolar range. We have developed a novel method to overcome these challenges, namely throughput and sensitivity. The novel Förster Resonance Energy Transfer (FRET)-Probe as an external probe is highly applicable to isothermal protein stability monitoring but also to conventional TSA. We have investigated ICD for multiple proteins with focus on KRASG12C with covalent inhibitors and three chemical denaturants performed at nanomolar protein concentration. Data showed corresponding inhibitor-induced stabilization of KRASG12C to those reported by nucleotide exchange assay.

Protein arginine N-methyltransferase 2 plays a noncatalytic role in the histone methylation activity of PRMT1.

Protein arginine N-methyltransferases are a family of epigenetic enzymes responsible for monomethylation or dimethylation of arginine residues on histones. Dysregulation of protein arginine N-methyltransferase activity can lead to aberrant gene expression and cancer. Recent studies have shown that PRMT2 expression and histone H3 methylation at arginine 8 are correlated with disease severity in glioblastoma multiforme, hepatocellular carcinoma, and renal cell carcinoma. In this study, we explore a noncatalytic mechanistic role for PRMT2 in histone methylation by investigating interactions between PRMT2, histone peptides and proteins, and other PRMTs using analytical and enzymatic approaches. We quantify interactions between PRMT2, peptide ligands, and PRMT1 in a cofactor- and domain-dependent manner using differential scanning fluorimetry. We found that PRMT2 modulates the substrate specificity of PRMT1. Using calf thymus histones as substrates, we saw that a 10-fold excess of PRMT2 promotes PRMT1 methylation of both histone H4 and histone H2A. We found equimolar or a 10-fold excess of PRMT2 to PRMT1 can improve the catalytic efficiency of PRMT1 towards individual histone substrates H2A, H3, and H4. We further evaluated the effects of PRMT2 towards PRMT1 on unmodified histone octamers and mononucleosomes and found marginal PRMT1 activity improvements in histone octamers but significantly greater methylation of mononucleosomes in the presence of 10-fold excess of PRMT2. This work reveals the ability of PRMT2 to serve a noncatalytic role through its SH3 domain in driving site-specific histone methylation marks.

Fluorometric detection of alpha-fetoprotein based on the use of a novel organic compound with AIE activity and aptamer-modified magnetic microparticles.

BACKGROUND: Liver cancer is one of the most common cancers in the world, and it seriously threatens human life and health. Alpha-fetoprotein (AFP), as a carcinogenic glycoprotein, is an important serum marker for detecting liver cancer. Therefore, the accurate and sensitive determination of AFP is crucial for the early diagnosis and treatment of liver cancer. To this end, a label-free fluorescence aptasensor for detecting AFP based on the use of a novel organic Compound D with an aggregation-induced emission activity and aptamer-modified magnetic microparticles was constructed. RESULTS: Compound D could combine with the complementary short chain of the aptamer (CSC-Apt) of AFP to form the D/CSC-Apt complex and realize the fluorescence enhancement of Compound D. Then, magnetic particles modified by the Apt of AFP (Apt-Fe3O4) were prepared. When AFP (or nontarget substance) and D/CSC-Apt were successively added to the Apt-Fe3O4 solution, Apt-Fe3O4 selectively bound to AFP or the D/CSC-Apt complex. Magnetic separation technology showed the changes in the fluorescence intensity of the supernatant. The research results revealed a good linear relationship between the changes in the fluorescence intensity of the supernatant and concentration of AFP within the concentration range of 10-10000 pg mL-1. The proposed aptasensor could achieve high-sensitivity and high-specificity detection of AFP, and its limit of detection was 3 pg mL-1 (S/N = 3). SIGNIFICANCE AND NOVELTY: The sensor combines the advantages of high selectivity of Apt, high sensitivity of fluorescence analysis, AIE effect and good water solubility of Compound D, and rapid separation using magnetic separation technology. And it can be directly used for the detection of AFP in actual serum samples with high accuracy, whereas most of the methods reported in the literature can only detect AFP in spiked serum samples.

Quantitation of autoinhibitory defects in pathogenic SHP2 mutants by differential scanning fluorimetry.

Src-homology-2-domain-containing protein tyrosine phosphatase-2 (SHP2) is a signaling enzyme whose activity is governed by an equilibrium between autoinhibited and activated states. Regulation of SHP2 activity is critical for cellular homeostasis, and mutations that alter its autoregulatory equilibrium cause cancers and developmental disorders. Several methods for assessing the strength of autoinhibitory interactions in SHP2 mutants have been previously reported, but each has limitations. We show that differential scanning fluorimetry provides a rapid, quantitative measure of SHP2 autoinhibition that is independent of the intrinsic activity of the SHP2 mutant being analyzed, does not involve protein labeling, and does not require specialized instrumentation.

Inorganic phosphate regulated high luminescence NaYF4:Yb3+, Er3+ as an iron ion fluorescent nanoprobe.

The iron ion in industrial circulating cooling water is an important indicator for early warning of equipment corrosion and control level. It is interesting to construct an upconversion luminescence iron ion nanoprobe with a common inorganic phosphate water treatment agent. Herein, inorganic phosphate sodium hexametaphosphate (SHMP) was used to regulate the morphology and functionalization of NaYF4:Yb3+, Er3+ upconversion luminescent nanoprobe (UCNPs) and applied to fluorometric detection of trace Fe(III) in water based on the fluorescence quenching which is caused by the selective coordination between hexametaphosphate on the surface of UCNPs and Fe(III). The structure, morphology, and luminous intensity of UCNPs were regulated by disodium hydrogen phosphate (ADSP), sodium tripolyphosphate (STPP) and sodium hexametaphosphate(SHMP). The UCNPs functionalized with SHMP has high sensitivity and selectivity for Fe(III) detection. The linear range and detection limit are 1.0-50 µM and 0.2 µM, respectively. The method has satisfactory results for the detection of trace Fe(III) in industrial circulating cooling water.

Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability.

Functional site-directed fluorometry has been the technique of choice to investigate the structure-function relationship of numerous membrane proteins, including voltage-gated ion channels. This approach has been used primarily in heterologous expression systems to simultaneously measure membrane currents, the electrical manifestation of the channels' activity, and fluorescence measurements, reporting local domain rearrangements. Functional site-directed fluorometry combines electrophysiology, molecular biology, chemistry, and fluorescence into a single wide-ranging technique that permits the study of real-time structural rearrangements and function through fluorescence and electrophysiology, respectively. Typically, this approach requires an engineered voltage-gated membrane channel that contains a cysteine that can be tested by a thiol-reactive fluorescent dye. Until recently, the thiol-reactive chemistry used for the site-directed fluorescent labeling of proteins was carried out exclusively in Xenopus oocytes and cell lines, restricting the scope of the approach to primary non-excitable cells. This report describes the applicability of functional site-directed fluorometry in adult skeletal muscle cells to study the early steps of excitation-contraction coupling, the process by which muscle fiber electrical depolarization is linked to the activation of muscle contraction. The present protocol describes the methodologies to design and transfect cysteine-engineered voltage-gated Ca2+ channels (CaV1.1) into muscle fibers of the flexor digitorum brevis of adult mice using in vivo electroporation and the subsequent steps required for functional site-directed fluorometry measurements. This approach can be adapted to study other ion channels and proteins. The use of functional site-directed fluorometry of mammalian muscle is particularly relevant to studying basic mechanisms of excitability.

Next Generation of Fluorometric Protease Assays: 7-Nitrobenz-2-oxa-1,3-diazol-4-yl-amides (NBD-Amides) as Class-Spanning Protease Substrates.

Fluorometric assays are one of the most frequently used methods in medicinal chemistry. Over the last 50 years, the reporter molecules for the detection of protease activity have evolved from first-generation colorimetric p-nitroanilides, through FRET substrates, and 7-amino-4-methyl coumarin (AMC)-based substrates. The aim of further substrate development is to increase sensitivity and reduce vulnerability to assay interferences. Herein, we describe a new generation of substrates for protease assays based on 7-nitrobenz-2-oxa-1,3-diazol-4-yl-amides (NBD-amides). In this study, we synthesized and tested substrates for 10 different proteases from the serine-, cysteine-, and metalloprotease classes. Enzyme- and substrate-specific parameters as well as the inhibitory activity of literature-known inhibitors confirmed their suitability for application in fluorometric assays. Hence, we were able to present NBD-based alternatives for common protease substrates. In conclusion, these NBD substrates are not only less susceptible to common assay interference, but they are also able to replace FRET-based substrates with the requirement of a prime site amino acid residue.

Review of Characteristics and Analytical Methods for Determination of Thiabendazole.

Thiabendazole (TBZ) is a fungicide and anthelmintic drug commonly found in food products. Due to its toxicity and potential carcinogenicity, its determination in various samples is important for public health. Different analytical methods can be used to determine the presence and concentration of TBZ in samples. Liquid chromatography (LC) and its subtypes, high-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC), are the most commonly used methods for TBZ determination representing 19%, 18%, and 18% of the described methods, respectively. Surface-enhanced Raman spectroscopy (SERS) and fluorimetry are two more methods widely used for TBZ determination, representing 13% and 12% of the described methods, respectively. In this review, a number of methods for TBZ determination are described, but due to their limitations, there is a high potential for the further improvement and development of each method in order to obtain a simple, precise, and accurate method that can be used for routine analysis.

Fluorimetric chemodosimeter for the detection of capsaicinoids in food matrices.

Capsaicin is a major pungent capsaicinoids in chili pepper and it causes duodenal, liver, stomach and gastric cancer in human. Hence, the detection of capsaicinoids becomes important on health issues concern. Here we are reporting, the first organic molecule based fluorimetric sensor for capsaicin detection using simple fluorophore 4-3-(pyren-2-yl-acryloyl) phenyboronic acid (PAPA), which was synthesized via greener microwave method. The probe has detected the capsaicin selectively in presence of other biomolecules in human biofluids through the intramolecular charge transfer mechanism and supported with DFT studies. The sensor has shown an excellent response towards capsaicin from 2 to 40 µM and the limit of detection of 12.84 nM. Real time analysis was done in various food matrices having capsaicinoids and the results have clearly shown good agreement with our optimized data and it also evinced that the developed sensor can be applied to detect the level of pungency of capsaicinoids.

Pyridinic-N-rich carbon dots in IFE-based Turn-off Fluorometric detection of nerve agent Mimic- Diethyl chlorophosphate and multicolor cell imaging.

Fluorometric sensors for the detection of nerve agent mimics have received a lot of interest nowadays due to their high sensitivity and selectivity, ease of operation, and real-time monitoring. Pyridinic-N-rich carbon dots (NCDs) prepared through microwave-assisted pyrolysis of l-Malic acid and urea have been explored first time in this work as a novel turn-off fluorescent probe for the sensitive and selective detection of diethyl chlorophosphate (DCP), a nerve agent mimic. The as-prepared carbon dots contained a large amount of pyridinic nitrogen on their surface, which can modulate the photoluminescence properties of the NCDs. The blue emissive NCDs possessed both excitation wavelength-dependent and independent emission behavior. The detection of DCP was premised on quenching of the fluorescence emission intensity of NCDs in the presence of similar chemical reagents (e.g., trimethyl phosphate, triethyl phosphate, triethyl phosphonoacetate, triphenyl phosphate, diphenyl phosphate, tributyl phosphate). Fluorescence quenching of the NCDs in the presence of DCP has been attributed to the inner filter effect (IFE). From the linear Stern-Volmer plot (R2 = 0.9992), the limit of detection (LOD) was found to be 84 µM for sensing DCP for the concentration ranging between 3 and 15 mM. The biocompatibility of NCDs was assessed through cytotoxicity assay on MDA-MB-231 breast cancer cells. Fluorescence imaging demonstrated that NCDs have low cytotoxicity and can be employed successfully in cell imaging.

Improved ANAP incorporation and VCF analysis reveal details of P2X7 current facilitation and a limited conformational interplay between ATP binding and the intracellular ballast domain.

The large intracellular C-terminus of the pro-inflammatory P2X7 ion channel receptor (P2X7R) is associated with diverse P2X7R-specific functions. Cryo-EM structures of the closed and ATP-bound open full-length P2X7R recently identified a membrane-associated anchoring domain, an open-state stabilizing "cap" domain, and a globular "ballast domain" containing GTP/GDP and dinuclear Zn2+-binding sites with unknown functions. To investigate protein dynamics during channel activation, we improved incorporation of the environment-sensitive fluorescent unnatural amino acid L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (ANAP) into Xenopus laevis oocyte-expressed P2X7Rs and performed voltage clamp fluorometry. While we confirmed predicted conformational changes within the extracellular and the transmembrane domains, only 3 out of 41 mutants containing ANAP in the C-terminal domain resulted in ATP-induced fluorescence changes. We conclude that the ballast domain functions rather independently from the extracellular ATP binding domain and might require activation by additional ligands and/or protein interactions. Novel tools to study these are presented.

Anthracene benzene conjugate (ABC): An asymmetric Schiff base for the selective detection of Ag+ ion using fluorimetry and its applications.

Anthracene based chemosensor ABC has been synthesized and characterized through 1H, 13C NMR, mass spectral studies. UV absorption and emission studies performed to identify the sensing behavior of chemosensor ABC. The probe ABC, originally bright fluorescent, selectively sense Ag+ ion by the quenching the fluorescence intensity through a "Switch On-off" process and quench the fluorescence due to the heavy atom effect interaction with the free chemosensor. The binding constant of the probe ABC with Ag+ was calculated as 5.4 × 104 M-1 and the limit of detection upto 1.4 nM level. The practical utilization of the probe ABC was demonstrated by applying to the real water and soil sample analysis, latent finger print, and the sensor as a fluorescent ink.

Chip-DSF: A rapid screening strategy for drug protein targets.

Identification of the drug target of lead compounds is an important means for rapid and efficient drug discovery. Protein chips are a high-throughput protein function analysis technology that has been widely used in screening drug protein targets in recent years. However, the verification of the results after high-throughput protein chip screening is still cumbersome. Based on our mature protein chip preparation platform, we prepared a protein chip containing 150 important high-frequency protein targets and used antibodies to prove the availability of the protein chip. To improve the accuracy of target screening, we combined the label-free differential scanning fluorimetry (DSF) with the protein chip, proposing the Chip-DSF strategy. Subsequently, we tested the method with small molecular ginsenoside-Rg2 (Rg2). The Chip-DSF strategy was used to successfully screen the potential target protein KRAS(G12C) of Rg2. Consistently, we found that Rg2 could inhibit NCI-H23 cell proliferation by inducing cell cycle arrest. Also, we found that Rg2 could reduce the amount of KRAS protein and inhibit the phosphorylation of KRAS downstream key signaling protein ERK1, RPS6, and P70S6K in NCI-H23 cells. Collectively, our Chip-DSF strategy could achieve rapid target verification which improved the accuracy and efficiency of target screening of protein chips.

A CuII -Salicylidene Glycinato Complex for the Selective Fluorometric Detection of Homocysteine over 20 Proteinogenic Amino Acids.

Homocysteine (Hcy) is a sulfur-containing α-amino acid that differs by one methylene (CH2 ) subunit from homologous cysteine (Cys). Elevated levels of Hcy are diagnostic markers of cardiovascular disease and other medical conditions. We present a new CuII -salicylidene glycinato complex 1 for the selective fluorometric detection of Hcy in water. In the presence of this analyte, the non-fluorescent copper-complex demetallates and disassembles into its building blocks. This process liberates a 3-chloro-5-sulfosalicylaldehyde signaling unit and is accompanied by a 51-fold turn-on fluorescence at 485 nm (λex =350 nm). Out of twenty proteinogenic amino acids, only histidine (12-fold turn-on fluorescence) and Cys (8-fold turn-on fluorescence) trigger some disassembly of probe 1. In comparison with important pioneering work on the detection of biothiols, this study strikingly demonstrates that structural modifications of chelate core structures steer substrate selectivity of metal-based probes. Importantly, probe 1 has proven suitable for the detection of Hcy in artificial urine.

Microtiter Plate-Based Differential Scanning Fluorimetry: A High-Throughput Method for Efficient Formulation Development.

Nano/microparticles are widely used as vaccine adjuvants to improve antigen stability and enhance immune response. Conformational stability of a given protein was normally assessed using differential scanning calorimetry (DSC) for the optimization of formulation and for ensuring antigen stability in vaccine products. Here, a higher throughput version, namely the microtiter plate-based differential scanning fluorimetry (DSF) method was developed and optimized for assessing the protein thermal stability in the particulate adjuvant-adsorbed form. Using recombinant human papillomavirus (HPV) vaccine antigens, along with several model proteins, enhanced sensitivity and correlation to the well-established differential scanning calorimetry were demonstrated. Higher throughput and much smaller sample consumption (1/10 ∼ 1/20 of the amount needed as compared to DSC) make the plate-based DSF a method of choice for formulation development, particularly during the early developmental phase of a project where the sample amount is usually quite limited.