Colorimetric Assay System Research Articles

In Situ Formed 3,3',5,5'-Tetramethylbenzidine-Cu(I/II) Charge-Transfer Complex Intermediates Promoting Colorimetric Assay of Cr6.

Developing a synthesis-free, multifunctional, and effective colorimetric assay system based on 3,3',5,5'-tetramethylbenzidine (TMB) oxidation is attractive yet challenging. Herein, we established a synergetic Cu2+/Cr6+-promoting strategy for TMB-based colorimetric detection of Cr6+. By introducing Cu2+, critical TMB·+···Cu(I/II)···TMB charge transfer complex (TMC) intermediates were in situ formed to reduce the activation energy of TMB oxidation, thereby accelerating Cr6+-mediated TMB oxidation. TMC intermediates also played a pivotal role in H2O2-participated TMB oxidation, clarifying the secondary responsibility of reactive oxygen species frequently caused by Fenton-like reactions. Leveraging the synergetic capacity between Cu2+ and Cr6+ for TMB oxidation, we demonstrated sensitive and specific colorimetric detections for Cr6+ with a limit of detection of 0.006 μM. With its convenient operation and rapid responsiveness, this strategy successfully enabled the practical detection of Cr6+ in real water samples. This work not only enhances the understanding of the internal acceleration mechanism in colorimetric sensing but also provides valuable opportunities to advance synthesis-free detection platforms.

Rapid detection of carbapenemase-producing gram-negative bacteria directly from blood culture bottles for a reliable guided empiric therapy.

Bloodstream infections (BSIs) caused by multidrug-resistant bacteria are a critical life-threatening challenge which necessitates the urgency to trigger life-saving treatment in a timely manner. This study aimed to evaluate the time required for rapid detection of carbapenemase-producing Enterobacterales (CPE) directly from blood culture bottles to optimize empirical treatment of BSI, especially in pediatric and infant patients, using a cost-effective method. This study included 419 Gram-negative bacteria, of which Klebsiella pneumoniae and Escherichia coli were the most common CPE causing BSI in pediatric and neonatal patients. Phenotypic and genotypic resistance of the selected isolates (45 K. pneumoniae and 9 E. coli) were determined by VITEK-2 Compact system and PCR, respectively. BACT/ALERT bottles were spiked with isolates. Finally, colorimetric RESIST-BC assay and Vitek-2 compact system were evaluated for the rapid detection of carbapenem-resistant bacteria directly from positive blood culture bottles. All selected isolates were phenotypically resistant to carbapenems. PCR showed that blaNDM and blaOXA-48 were present in all isolates, blaVIM was present in 44.4%, while blaKPC and blaIMP were entirely absent. The RESIST-BC kit showed good agreement with PCR for blaNDM and blaOXA-48, demonstrating high sensitivity and specificity, but not with blaVIM. These findings point out that RESIST-BC assay demonstrated an exceptionally short detection time for CPE, completing all cases within the first hour after the blood culture bottles flagged positive. It is also superior in providing a cluefor clinicians on antibiotic combinations that can be administered, depending on the type of β-lactamases detected, promptly and efficiently, with low expenses.

Label-free colorimetric analysis strategies based on adsorption-responsive surface-enhanced photochromic phenomena of tungsten(VI) oxide nanoparticles for amino acids.

Herein, we present a colorimetric detection method based on the surface-enhanced photochromic phenomenon of tungsten (VI) oxide (WO3) nanocolloid particles for α-amino acid (AA) molecules, including L-aspartic acid (Asp), L-glutamic acid (Glu), L-histidine (His), L-isoleucine (Ile), L-leucine (Leu), L-lysine (Lys), L-phenylalanine (Phe), and L-valine (Val). The UV-induced photochromic phenomena in the AA/WO3 binary aqueous systems were investigated using UV-Vis absorption spectrometry. The adsorption properties of the AA molecules on the surface of the WO3 nanocolloid particles have been identified using a combination of adsorption isotherm analysis and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. A good linear correlation between the concentration of the AAs adsorbed on the surface of the WO3 nanocolloid particles and the initial photochromic coloration rate in the corresponding UV-irradiated WO3 colloidal aqueous solution was obtained with over three orders of magnitude, indicating that the surface-enhanced photochromic phenomenon of the WO3 nanocolloid particle can be used to detect the AA molecules. In addition, based on the results of the UV-Vis absorption, ATR-FTIR, and adsorption isotherm analyses, we have experimentally demonstrated that the AA/WO3 binary aqueous system with inner-sphere adsorbed Ile, Leu, Lys, or Val molecules on the surface of the WO3 nanocolloid particles exhibits a more significant surface-enhanced photochromic phenomenon than the system with outer-sphere adsorbed Asp, Glu, His, or Phe molecules. The strong inner-sphere adsorption of the AA molecules successfully improved the limit of detection. This study provides valuable insights into a "label-free" colorimetric assay system based on the surface-enhanced photochromic phenomenon of the WO3 nanocolloid probe.

Novel Tetrazolium-Based Colorimetric Assay for Helicase nsp13 in SARS-CoV-2

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a human pathogenic virus that encodes for a helicase (SC2Hel) that is essential for viral replication. SC2Hel has the ability to unravel dsRNA or dsDNA in an NTP-dependent manner from the 5′ to 3′ directionality. The standard helicase assay from studies involving SARS-CoV and SARS-CoV-2 have relied on the concept of fluorescence resonance energy transfer. Adding to the collection of helicase assays, herein, we have developed a novel tetrazolium-based colorimetric assay system for the detection of ADP that is produced via SC2Hel activity. This SC2Hel assay combines three enzyme-coupled steps involving the ADP-dependent Thermococcus litoralis glucokinase (TlGlcK), Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (LmG6PDH), and Clostridium kluyveri diaphorase (CkDIA). Iodonitrotetrazolium chloride (INT), a colorimetric tetrazolium reagent, was used in the final step of the assay that converted into INT-formazan during reduction. INT-formazan in the assay’s buffered solution at pH 7.6 exhibited an intense colorimetric response at a wavelength maximum of 505 nm. The assay exhibited excellent performance characteristics as it revealed a Z’ factor of 0.87 and it has the potential to be further adopted into high-throughput screening studies for therapeutic drug discovery research.

Cyclodextrin-Assisted Surface-Enhanced Photochromic Phenomena of Tungsten(VI) Oxide Nanoparticles for Label-Free Colorimetric Detection of Phenylalanine.

Herein are presented the results of experiments designed to evaluate the effectiveness of host-guest interactions in improving the sensitivity of colorimetric detection based on surface-enhanced photochromic phenomena of tungsten(VI) oxide (WO3) nanocolloid particles. The UV-induced photochromic coloration of WO3 nanocolloid particles in the presence of aromatic α-amino acid (AA), l-phenylalanine (Phe) or l-2-phenylglycine (Phg), and heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin (TMβCDx) in an aqueous system was investigated using UV-vis absorption spectrometry. The characteristics of the adsorption modes and configurations of AAs on the WO3 surface have also been identified by using a combination of adsorption isotherm analysis and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). A distinct linear relationship was observed between the concentration of AAs adsorbed on the WO3 nanocolloid particles and the initial photochromic coloration rate in the corresponding UV-irradiated colloidal WO3 in aqueous media, indicating that a simple and sensitive quantification of AAs can be achieved from UV-induced WO3 photochromic coloration without any complicated preprocessing. The proposed colorimetric assay in the Phe/TMβCDx/WO3 ternary aqueous system had a linear range of 1 × 10-8 to 1 × 10-4 mol dm-3 for Phe detection, with a limit of detection of 8.3 × 10-9 mol dm-3. The combined results from UV-vis absorption, ATR-FTIR, and adsorption isotherm experiments conclusively indicated that the TMβCDx-complexed Phe molecules in the Phe/TMβCDx/WO3 ternary aqueous system are preferentially and strongly inner-sphere adsorbed on the WO3 surface, resulting in a more significant surface-enhanced photochromic phenomenon. The findings in this study provided intriguing insights into the design and development of the "label-free" colorimetric assay system based on the surface-enhanced photochromic phenomenon of the WO3 nanocolloid probe.

Design of CsPbBr3 NCs/MnO2 based sensing system for multi-mode detection of D-penicillamine

Herein, a multi-mode assay platform was constructed for the detection of D-penicillamine based on perovskite nanocrystals (CsPbBr3 NCs) and MnO2 nanosheets. The water-dispersible CsPbBr3 NCs synthesized via aqueous emulsion process has a strong fluorescence emission at 520 nm. The electrostatic interaction between positively charged CsPbBr3 NCs and negatively charged MnO2 nanosheets would lead to the fluorescence quenching of CsPbBr3 NCs on account of the inner filter effect (IFE). The addition of D-penicillamine could convert MnO2 nanosheets to Mn2+ and weaken inner filter effect, resulting in distinct recovery of fluorescence at 520 nm and the decrease of the absorption of CsPbBr3 NCs/MnO2 at 375 nm. Therefore, the fluorescence and colorimetric assay system for D-penicillamine was developed. The fluorescence and colorimetric sensing system could detect D-penicillamine ranging from 0.1 to 40 µM and 0.5 to 40 µM with the limits of detection (LOD) were 0.061 µM and 0.24 µM, respectively. The developed assay system could be applied to analyze D-penicillamine in real samples and acquired satisfactory results. Furthermore, a portable intelligent detection device was developed based on the mobile phone and RGB analysis software, demonstrating the practicability and potential of the sensing system.

Application of Peroxidase-Mimic Mn2BPMP Boosted by ADP to Enzyme Cascade Assay for Glucose and Cholesterol

The Mn2BPMP complex has an intrinsic peroxidase-like activity in the pH range of 5 to 8, especially a maximum activity at pH 7, while most peroxidase mimics operate at an acidic pH (mainly pH 4). Its peroxidase-like activity is high among small-molecule-based peroxidase mimics with a high reproducibility. In addition, we recently revealed that adenosine mono/diphosphate (AMP and ADP) significantly boosted the peroxidase-like activity of Mn2BPMP. These advantages imply that Mn2BPMP is suitable for biosensing as a substitute for horseradish peroxidase (HRP). Herein, we established a colorimetric one-pot assay system using the enzyme cascade reaction between analyte oxidase and ADP-boosted Mn2BPMP. The simple addition of ADP to the Mn2BPMP-based assay system caused a greater increase in absorbance for the same concentration of H2O2, which resulted in a higher sensitivity. It was applied to one-pot detection of glucose and cholesterol at 25 °C and pH 7.0 for a few minutes.

Colorimetric detection of acetylcholinesterase and its inhibitor based on thiol-regulated oxidase-like activity of 2D palladium square nanoplates on reduced graphene oxide.

Aconvenient and sensitive colorimetricassay for acetylcholinesterase (AChE) and its inhibitor has been designedbased on the oxidase-like activity of {100}-faceted Pd square nanoplates which are grown in situ on reduced graphene oxide (PdSP@rGO). PdSP@rGO can effectively catalyze the oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) without the assistance of H2O2 to generate blue oxidized TMB (oxTMB) with a characteristic absorption peak at 652nm. In the presence of AChE, acetylthiocholine (ATCh), a typical AChE substrate, is hydrolyzed to thiocholine (TCh). The generated TCh can effectively inhibit the PdSP@rGO-triggered chromogenic reaction of TMB via cheating with Pd, resulting in color fading and decrease in absorbance. Thus, a sensitive probe for AChE activity is constructed with a working range of 0.25-5mUmL-1 and a limit of detection (LOD) of 0.0625mUmL-1. Furthermore, because of the inhibition effect of tacrine on AChE, tacrine is also detected through the colorimetric AChE assay system within the concentrations range 0.025-0.4μM with a LOD of 0.00229μM. Hence, a rapid and facile colorimetric procedure to sensitively detect AChE and its inhibitor can be anticipated through modulating the oxidase-like activity of PdSP@rGO. Colorimetric method for detection of AChE and its inhibitor is established by modulating the oxidase mimetic activity of {100}-faceted Pd square nanoplates on reduced graphene oxide (PdSP@rGO).

Organic Nanocrystal Coated-Microfluidic Paper Analytical Device for Heavy Metal Ions Detection

Introduction Monitoring heavy metal ions in the environment especially in river water is very important due to their high toxicity. Heavy metal ions can accumulate in the water and the excessive heavy metal intake become serious problem causing many negative impacts on human health such as kidney, liver, and lung damage. Many techniques are available to analyze the total amount of heavy metal ions in the water, but most techniques require high cost, samples pretreatment and expert operator, such as inductively coupled plasma mass spectrometry (ICP-MS), atomic absorption spectroscopy (AAS), atomic emission spectroscopy (AES), UV-Vis spectroscopy, X-ray fluorescence and anodic stripping voltammetry.Low cost, simple and reliable technologies are preferred for determination of heavy metal ions without reducing analytical precision. Microfluidic paper analytical device (µPADs) is one of the most promising platforms in many fields especially in heavy metal ion analysis which satisfy these requirements. Selection of µPADs to determine heavy metal ions in river water is due to its cheapness, ease of use, and simplicity. Here, we combined µPADs with organic nanocrystal to enhance the sensitivity of heavy metal detection.In this work, we developed for the first time organic nanocrystal coated µPADs which are effective for the determination of heavy metal ions. Simple re-precipitation method was used for generation of organic nanocrystals (NCs) composed of hydrophobic chelating agents [1,2]. NCs were coated uniformly over paper surface and could not be easily removed by employing aqueous metal-ion solution. In addition, organic nanocrystal has chemical response inherited from the chelate and is good for sorption-based color generation. The color changes of organic nanocrystal coated µPADs after addition of metal solutions were analyzed. Method Organic nanocrystals were prepared by injection of hydrophobic chelating agent solution into aqueous buffer [tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS) at pH 8.4 under a high speed stirring condition. µPADs were fabricated by a photolithographic method [3] with minor modification using SU-8 3050 as photoresist. NCs solution (750 µL) was introduced to µPADs with a micropipette with an injection rate of 25 µL /s, and then it was dried under desiccator vacuum for 30 minutes. Colorimetric method has been used for quantitative analysis from multiple color changes based on the L*a*b* color space [4]. A digital image of the NCs coated-μPADs was taken with a smartphone (Samsung J5) before and after exposing to a metal solution under a light box. The distance between NCs coated-μPADs and the smartphone camera was 12 cm. The L*a*b* images were obtained with ImageJ software. Results and Discussions NCs of three organic chelating reagents, bathophenanthroline (Bathophen), 1-(2-pyridylazo)-2-naphthol (PAN), and 1-(2-thiazolylazo)-2-naphthol (TAN) have been successfully coated onto µPADs. Scanning electron microscope (SEM) observation revealed that organic nanocrystals were not removed by exposure to excessive metal solutions. Organic nanocrystal coated-µPADs have a strong response to heavy metal ion solution at ppm level and showed different color with different heavy metal ions.Multivariate data analysis, with 3-dye NCs color-responses to several ion species, was utilized to determine the concentration of heavy metal ions in river water. 250 µL heavy-metal-ions solution in ppb level was applied to NCs-coated µPADs. The results showed that NCs-coated µPADs is a promising device for heavy metal ions determination in river water. In the presentation, we will report the preparation method of NCs-coated µPADs and its application to heavy metal ions determination. Acknowledgements This work was supported by the Research Program of "Five-star Alliance" in "NJRC Mater. & Dev." References [1] Kasai, H. Nalwa, H. S., Oikawa, H., Okada, S., Matsuda, H., Minami, N., Kakuta, A., Ono, K., Mukoh, A., and Nakanishi, H., A Novel Preparation Method of Organic Microcrystals, Jpn. J. Appl. Phys., 31 (8), L1132-4 (1992).[2] Takahashi,Y., Kasai,H., Nakanishi, H., and Suzuki, TM. Test Strips for Heavy-Metal Ions Fabricated from Nanosized Dye Compounds, Angew. Chem. Int. Ed. 45, 913–916 (2006) DOI: 10.1002/anie.200503015[3] Busa, L.S.A., Maeki,M., Ishida, A., Tanib, H. and Tokeshi, M.,Simple and Sensistive Colorimetric Assay System For Horseradish Peroxidase Using Microfluidic Paper-Based Devices, Sensors and Actuators B 236 433-441 (2016). http://dx.doi.org/10.1016/j.snb.2016.06.013[4] Komatsu,T. Mohammadi, S., Busa, L.S.A., Maeki,M., Ishida, A., Tanib, H. and Tokeshi, M. Image Analysis for a Microfluidic Paper-Based Analytical Device Using The CIE L*A*B* Color System, Analyst, 141, 6507 (2016) DOI: 10.1039/c6an01409g

A paper-based colorimetric assay system for sensitive cysteine detection using a fluorescent probe.

A fluorescent probe for the colorimetric detection of cysteine (Cys) was synthesized by combining resorufin with 7-nitrobenzofurazan. The resultant probe was characterized by FT-IR spectroscopy, fluorescence detection, mass spectrometry and NMR spectroscopy. After reacting with Cys, resorufin is released and the color changes from light yellow to red on paper-based analytical devices. The results were recorded using a common cell phone and subsequently analyzed using Photoshop software. The color intensity of the RGB channel increased linearly in the 0.04 to 70.04 μM Cys concentration range when the probe concentration was 2.6 mM (R = 0.9965), and the limit of detection was 16 nM. The effects of detection conditions have been investigated and are discussed. The interference deviations of 13 substances to Cys were in the range of -2% to +2%. The Cys concentration was determined as 270.8 ± 19.3 μM in human plasma, and the recovery was from 99.7% to 100.4%. The work demonstrates the potential of the method to detect Cys in real samples with low cost and high sensitivity.

A colorimetric assay system for dopamine using microfluidic paper-based analytical devices

We report the colorimetric detection of dopamine (DA) on microfluidic paper-based analytical devices (μPADs) using an oxidation-reduction method. Here, dopamine reacts with ferric chloride forming reduced Fe2+ that subsequently reacts with phenanthroline to form the red tris(1,10-phenanthroline)iron(II) complex. The devices were fabricated by wax printing and changes in color intensity were recorded using a common cell phone. Subsequent analysis using Photoshop software, yielded a limit of detection (LOD) for DA of 0.37 μmol/L with a linear range of 0.527–4.75 μmol/L and relative standard deviation of 0.11% (inter-day) and 0.15% (intra-day) for n = 15 paper chips. The effects of detection conditions have been investigated and are discussed. Cow serum samples and human blood serum and plasma samples were detected. The work, herein, demonstrates the potential of this method as a low cost and rapid colorimetric technique to detect DA in real samples.

MiR-26a inhibits atherosclerosis progression by targeting TRPC3

BackgroundAtherosclerosis, a chronic multi-factorial vascular disease, has become a predominant cause of a variety of cardiovascular disorders. miR-26a was previously reported to be involved in atherosclerosis progression. However, the underlying mechanism of miR-26a in atherosclerosis remains to be further explained.MethodsHigh-fat diet (HFD)-fed apolipoprotein E (apoE)−/− mice and oxidized low-density lipoprotein (ox-LDL)-stimulated human aortic endothelial cells (HAECs) were established as in vivo and in vitro models of atherosclerosis. RT-qPCR and western blot analysis were performed to measure the expression of miR-26a and transient receptor potential canonical 3 (TRPC3), respectively. Binding between miR-26a and TRPC3 was predicted with bioinformatics software and verified using a dual luciferase reporter assay. The effects of miR-26a on the lipid accumulation, atherosclerotic lesion, and inflammatory response in HFD-fed apoE−/− mice were investigated by a colorimetric enzymatic assay system, hematoxylin–eosin and oil-Red-O staining, and ELISA, respectively. Additionally, the effects of miR-26a or combined with TRPC3 on cell viability, apoptosis and the nuclear factor-kappa B (NF-κB) pathway in ox-LDL-stimulated HAECs were evaluated by MTT assay, TUNEL assay, and western blot, respectively.ResultsmiR-26a was downregulated in HFD-fed apoE−/− mice and ox-LDL-stimulated HAECs. miR-26a overexpression inhibited the pathogenesis of atherosclerosis by attenuating hyperlipidemia, atherosclerotic lesion and suppressing inflammatory response in HFD-fed apoE−/− mice. Moreover, miR-26a overexpression suppressed inflammatory response and the NF-κB pathway, promoted cell viability and inhibited apoptosis in ox-LDL-stimulated HAECs. Additionally, TRPC3 was demonstrated to be a direct target of miR-26a. Enforced expression of TRPC3 reversed the effects of miR-26a on cell viability, apoptosis, and the NF-κB pathway in ox-LDL-treated HAECs.ConclusionsmiR-26a alleviated the development of atherosclerosis by regulating TRPC3, providing a potential target for atherosclerosis treatment.

A sensitive colorimetric assay system for nucleic acid detection based on isothermal signal amplification technology.

Rapid and accurate detection of microRNAs in biological systems is of great importance. Here, we report the development of a visual colorimetric assay which possesses the high amplification capabilities and high selectivity of the rolling circle amplification (RCA) method and the simplicity and convenience of gold nanoparticles used as a signal indicator. The designed padlock probe recognizes the target miRNA and is circularized, and then acts as the template to extend the target miRNA into a long single-stranded nucleotide chain of many tandem repeats of nucleotide sequences. Next, the RCA product is hybridized with oligonucleotides tagged onto gold nanoparticles. This interaction leads to the aggregation of gold nanoparticles, and the color of the system changes from wine red to dark blue according to the abundance of miRNA. A linear correlation between fluorescence and target oligonucleotide content was obtained in the range 0.3-300 pM, along with a detection limit of 0.13 pM (n=7) and a RSD of 3.9% (30 pM, n=9). The present approach provides a simple, rapid, and accurate visual colorimetric assay that allows sensitive biodetection and bioanalysis of DNA and RNA nucleotides of interest in biologically important samples. Graphical abstract The colorimetric assay system for analyzing target oligonucleotides.

Ultra-Sensitive Colorimetric Assay System Based on the Hybridization Chain Reaction-Triggered Enzyme Cascade Amplification.

A versatile and ultrasensitive colorimetric detection platform has been developed based on the hybridization chain reaction (HCR)-triggered enzyme cascade amplification in this work. The proposal involves the preparation of two different hairpin DNA strands consisting of the H1, modified with glucose oxidase (GOx-H1) and H2, modified with horseradish peroxidase (HRP-H2). The H1 and H2 were composed of complementary sequence of nucleic acid target (T) and interlaced complementary stem-loop sequences. In the nucleic acid detection, the hybridization of T and its complementary sequence induces the autonomous assembly of GOx-H1 and HRP-H2 through the predictable HCR, accompanied by the formation of GOx/HRP enzyme pairs with a multiple enzymatic cascade. In contrast to the crude mixture of free GOx-H1 and HRP-H2, the catalytic performance of enzyme cascade reaction has been significantly enhanced, which can be determined by monitoring the absorbance change of 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS2-), a typical substrate with hydrogen peroxide for the HRP. Furthermore, this platform can be utilized in the assay of biological substances by the introduction of corresponding aptamer (Apt), complementary strands (Com), and an assistant hairpin DNA strand (HAssist). In view of the signal amplification of HCR and the enhanced catalytic performance of cascaded enzymes, our colorimetric assay system exhibits excellent sensitivity, and the detection limits have been calculated to be 5.2 fM and 0.8 pM for the nucleic acid target (T as a model) and biological substances (ATP as a model), respectively.

Simple and sensitive colorimetric assay system for horseradish peroxidase using microfluidic paper-based devices

This article describes the development of a simple, portable assay system using microfluidic paper-based analytical devices (μPADs) coupled with colorimetric detection for rapid measurements. The properties of different paper substrates were first investigated to determine which type of paper would be the most suitable for the fabrication of the μPADs. Simultaneous detection of horseradish peroxidase (HRP) utilizing a 5μL sample analytical volume was demonstrated using a single μPAD. Hydrophilic test regions were separated by hydrophobic barriers, which were fabricated through photolithography. These test regions were immobilized with 10mM of 3,3′,5,5′-tetramethylbenzidine for HRP assay. The detection range obtained with the proposed system covered HRP concentrations from 0.37 to 124fmol (or 3–1000ngmL−1). The detection limit (blank+3σ) for HRP was calculated to be 0.69 fmol (or 5.58ngmL−1) through a 4-parameter logistic nonlinear regression using results obtained within a 15min assay time. The findings obtained using the developed system suggest that μPAD assay systems for simple but highly sensitive measurements can be designed to give on-site determinations of target compounds using peroxidase-conjugated molecules.

Guanine-rich DNA-based peroxidase mimetics for colorimetric assays of alkaline phosphatase.

DNA-based peroxidase mimetics are facilely constructed through Cu(II)-coordination with different oligonucleotides involving G20, C20, A20 and T20, respectively, with high peroxidase mimicking activity as well as high stability against proteins. Peroxidase-like activities of DNA-Cu(II) complexes are greatly associated with the sequence composition of DNA templates, which decrease in the following order: G20>C20>A20>T20. G20-Cu(II) complex ([Cu(2+)]/[base]=0.05) possesses the Km value of 0.257 mM toward 3,3',5,5'-tetramethylbenzidine and 102.3mM toward hydrogen peroxide at 25 °C. G20-Cu(II) complexes are employed to develop a colorimetric turn-on assay of alkaline phosphatase with high sensitivity and selectivity, on the basis of pyrophosphate-induced inhibition of their intrinsic peroxidase-like activities. The limit of detection is achieved as 0.84 U/L with the linear response region of 20-200 U/L. Such colorimetric assay system is probably applicable for the quantitative determination of ALP in biological fluids.

Spectrophotometric assay of D-isoleucine using an artificially created D-amino acid dehydrogenase.

D-isoleucine (D-Ile) can be assayed using chiral chromatography but the availability of that method is limited by the necessity for special expertise and expensive equipment. We therefore developed a simple and specific colorimetric assay system for D-Ile determination using an artificially created NADP(+)-dependent D-amino acid dehydrogenase (DAADH). The system consists of two reaction steps: the first is the quantitative conversion of D-Ile to (3R)-2-oxo-3-methyl valerate by DAADH in which NADP(+) is converted to NADPH, while the second is chemical conversion of NADPH to reduced water-soluble Tetrazolium-3 via a redox mediator. D-Ile was determined from 1 to 50µM, and the assay was unaffected by the presence of any of three other isomers (100µM), alcohol and organic acids.

Gold nanoparticle-based colorimetric chiral discrimination of histidine: application to determining the enantiomeric excess of histidine

The proposed colorimetric assay system consisting of L-Pro group-functionalized gold nanoparticles (L-Pro–AuNPs) and Cu2+ ions can discriminate the absolute configuration of His and determine the enantiomeric excess of His.

Discovery of a new sesquiterpenoid from Phellinus ignarius with antiviral activity against influenza virus

Seven natural products, including one new sesquiterpenoid (eudesm-1β, 6α, 11-triol, compound 1), one ergosta -4, 6, 8(14), 22-tetraen-3-one (compound 2), four polyphenols (compounds 3, 4, 5, 6), and one pyrone (3-hydroxy-2-methyl-4-pyrone, compound 7) were isolated from cultures of Phellinus ignarius by column chromatography. The detailed structure of compound 1 was determined using a combination of one- and two-dimensional nuclear magnetic resonance, mass spectrometry and infrared spectroscopy. The antiviral activity of these compounds against H5N1 influenza A virus was investigated using an MTT colorimetric assay system in Madin-Darby canine kidney cells. The results indicate that compound 1 possesses significant ability to inhibit influenza virus. The 50% effective concentration was 0.14 ± 0.04 μM. Molecular modeling further suggested that the anti-influenza virus activity of this compound was partially attributed to the interactions of hydroxyl groups with an amino acid residue (Asn 170) of neuraminidase (NA) at the binding site. Moreover, the results of enzyme inhibition assays indicated that 50% inhibition of NA was achieved by compound 1 at a concentration of 0.657 ± 0.325 mg/mL, which suggested that compound 1 is likely to interact with the NA enzyme.

MMP-2 assay within the hybrid layer created by a two-step etch-and-rinse adhesive: Biochemical and immunohistochemical analysis

ObjectiveDegradation of hybrid layers (HLs) within resin-infiltrated dentine results from multiple degradation factors, including collagenolytic activity of specific matrix metalloproteinases (MMPs). Inhibition of host-derived MMPs may, therefore, slow the degradation of HL. The null hypothesis tested is that the presence of MMP-2 is similar regardless of chlorhexidine (CHX) pre-treatment or the use of an adhesive. MethodsPowdered dentine prepared from extracted human teeth was divided into 4 groups: (G1) mineralised powder (control group); (G2) dentine powder treated with 1% phosphoric acid for 1min; (G3) 1% phosphoric acid-etched dentine treated with Adper Scotchbond 1 XT (SB1XT; 3M ESPE); (G4) 1% phosphoric acid-etched dentine treated with 0.2% CHX followed by SB1XT. The concentration of detectable pro-MMP-2 and MMP-2 was assayed using a colorimetric assay system (QuantiSir). In addition, the presence of MMP-2 in the HL was assessed in 1 year-aged adhesive–dentine interfaces using an immunohistochemical approach under FEI-SEM/TEM. ResultsIn dentine powder treated with 1% phosphoric acid (G2), MMP-2 level decreased compared to controls (G1); the application of SB1XT (G3) resulted in an increase of MMP-2, whilst 0.2% CHX before SB1XT application (G4), reduced MMP-2. The FEI-SEM/TEM analysis revealed MMP-2 distribution within the HL of aged interfaces showing increase MMP-2 patterns in the control group and minor labelling in the CHX-pretreated specimens. ConclusionThe results of this study support the use of non-toxic MMPs inhibitors, such as CHX, as an appropriate additional step in bonding procedures in order to increase the longevity of the adhesive restorations.