Peroxidyme‐Amplified Radical Chain Reaction (PARCR): Visible Detection of a Catalytic Reporter

Peroxidyme Amplified Radical Chain Reaction (PARCR), a novel enzyme‐free system that achieves exponential amplification of a visible signal, is presented. Typical enzyme‐free amplification systems that produce a visible readout suffer from long reaction times, low sensitivity, and narrow dynamic range. PARCR employs photocatalyzed nonlinear signal generation, enabling unprecedented one‐pot, naked‐eye detection of a catalytic reporter from 1 μm down to 100 pm . In this reaction, hemin‐binding peroxidase‐mimicking DNAzymes (“peroxidymes”) mediate the NADH‐driven oxidation of a colorless, nonfluorescent phenoxazine dye (Amplex Red) to a brightly colored, strongly fluorescent product (resorufin); illumination with green light initiates multiple radical‐forming positive‐feedback loops, rapidly producing visible levels of resorufin. Collectively, these results demonstrate the potential of PARCR as an easy‐to‐use readout for a range of detection schemes, including aptamer labels, hybridization assays, and nucleic acid amplification.     

Improvement in the sensitivity of microfluidic ELISA through field amplified stacking of the enzyme reaction product

In this article, we demonstrate a novel approach to enhancing the sensitivity of enzyme-linked immunosorbent assays (ELISA) through pre-concentration of the enzyme reaction product (resorufin/4-methylumbelliferone) in free solution. The reported pre-concentration was accomplished by transporting the resorufin/4-methylumbelliferone molecules produced in the ELISA process towards a high ionic-strength buffer stream in a microfluidic channel while applying a voltage drop across this merging region. A sharp change in the electric field around the junction of the two liquid streams was observed to abruptly slow down the negatively charged resorufin/4-methylumbelliferone species leading to the reported pre-concentration effect based on the field amplified stacking (FAS) technique. It has been shown that the resulting enhancement in the detectability of the enzyme reaction product significantly improves the signal-to-noise ratio in the system thereby reducing the smallest detectable analyte concentration in the ELISA method. Applying the above-described approach, we were able to detect mouse anti-BSA and human TNF-α at concentrations nearly 60-fold smaller than that possible on commercial microwell plates. For the human TNF-α sample, this improvement in assay sensitivity corresponded to a limit of detection (LOD) of 0.102 pg mL⁻¹ using the FAS based microfluidic ELISA method as compared to 7.03 pg mL⁻¹ obtained with the traditional microwell plate based approach. Moreover, because our ELISAs were performed in micrometer sized channels, they required sample volumes about two orders of magnitude smaller than that consumed in the latter case (1 μL versus 100 μL).     

Multiplex ELISA in a single microfluidic channel

In this article, we demonstrate a novel approach to implementing multiplex enzyme-linked immunosorbent assay (ELISA) in a single microfluidic channel by exploiting the slow diffusion of the soluble enzyme reaction product across the different assay segments. The functionality of the reported device is realized by creating an array of ELISA regions within a straight conduit that are selectively patterned with chosen antibodies/antigens via a flow-based method. The different analytes are then captured in their respective assay segments by incubating a 5-μL aliquot of sample in the analysis channel for an hour under flow conditions. Once the ELISA surfaces have been prepared and the enzyme substrate introduced into the analysis channel, it is observed that the concentration of the soluble enzyme reaction product (resorufin) at the center of each assay region grows linearly with time. Further, the rate of resorufin generation at these locations is found to be proportional to the concentration of the analyte being assayed in that segment provided that the ELISA reaction time in the system (τ _R ) is kept much shorter than that required by the resorufin molecules to diffuse across an assay segment (τ _D ). Under the operating condition τ _R  << τ _D , the reported device has been shown to have a 35% lower limit of detection for the target analyte concentration compared with that on a commercial microtiter plate using only a twentieth of the sample volume.     

Fluorometric Determination of Cellulase

Abstract

A fluorometric procedure is described for the determination of the enzyme cellulase. The method is based upon the hydrolysis of the nonfluorescent substrate, resorufin acetate, by the enzyme to give the highly fluorescent resorufin (λ_ex = 540 mμ;, λ_em = 580 mμ). By this procedure from 0.00010 to 0.060 units per ml. of cellulase can be determined with an accuracy and deviation of about 1.5%. Evidence is offered to demonstrate cellulase, and not esterase, activity.     

A spectroscopic off-on probe for simple and sensitive detection of carboxylesterase activity and its application to cell imaging

A new resorufin-based spectroscopic probe, 7-(p-acetoxyphenylmethyloxy)-3H-phenoxazin-3-one (1), has been developed for detecting carboxylesterase activity. The probe is designed by introducing p-acetoxyphenylmethyloxy as a bifunctional moiety to resorufin. The p-acetoxyphenylmethyloxy moiety not only quenches the spectroscopic signal of resorufin, but also serves as a recognition unit for carboxylesterase. As a result, the prepared latent spectroscopic probe 1 shows very low background signal, which is rather desired for achieving sensitive detection. The specific cleavage of the carboxylic ester bond by carboxylesterase induces the hydrolysis of the probe, resulting in the release of resorufin and thereby the recovery of both color and fluorescence signal. This behaviour leads to the development of a simple and sensitive fluorescent method for assaying carboxylesterase activity, with a detection limit of 8.6 × 10(-5) U mL(-1), which is much more sensitive than the existing fluorescence approaches. Moreover, experimental results showed that the probe 1 is cell membrane permeable, and its applicability has been demonstrated for monitoring carboxylesterase activity in HeLa cells.     

Lead determination at the ppb level using stopped-flow FIA

A flow-injection-based kinetic method for the determination of lead ions was developed. In this method, resazurin was reduced to resorufin by sulfide and lead ions. The method has a detection limit of 1 ppb with a relative standard deviation of 5.2% at the 20 ppb level. Interferences can be removed by prior extraction.     

β-葡萄糖醛酸苷酶荧光底物试卤灵葡萄糖醛酸苷的高效制备

以猴肝微粒体(CyLM)为酶源, 采用生物制备法实现了荧光底物试卤灵(Resorufin)向试卤灵葡萄糖醛酸苷(Resorufin β-D-glucuronide)的高效转化, 同时借助新型色谱分离材料C18WAX及固相萃取技术实现了Resorufin β-D-glucuronide的高效富集及选择性洗脱, 最终获得纯度大于98%的目标产物. 所得产物结构经LC-MS, ¹H NMR和¹³C NMR等手段进行了表征. 在此基础上, 以该葡萄糖醛酸产物为探针底物建立了β-葡萄糖醛酸苷酶活性检测及抑制剂高通量筛选的方法.     

A naked-eye visible and fluorescence "turn-on" probe for acetyl-cholinesterase assay and thiols as well as imaging of living cells

A resorufin derivative with a DBS group (probe 1) was designed and investigated for the detection of acetylcholinesterase (AChE) and inhibitor screening. The new assay is based on cascade enzymatic and chemical reactions of ATC, AChE and probe 1, and it can be carried out in a dual-signal detection mode. Moreover, the results show that probe 1 can be used for cell fluorescence staining.     

Nanophotonic Device in Combination with Bacteriophages for Enhancing Detection Sensitivity of Escherichia coli in Simulated Wash Water

Among various approaches to control and monitor cross-contamination of fresh produce, a biosensor that can rapidly detect the presence of a specific bacterium in wash water or on fresh produce can be effective. This research demonstrates the development of a rapid biosensor based on a combination of a nanophotonic device and the bacteriophage T7 for the detection of Escherichia coli without the need for culturing or nucleic acid extraction. This biosensor platform is based on bacteriophage mediated specific lysis of target bacteria and release of β-galactosidase. The enzyme could be further detected by a nanophotonic device that amplifies the fluorescent signal, therefore allowing better sensitivity. Production of β-galactosidase is induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) and the enzyme is then released by bacteriophage lysis, which is detected by the nanophotonic device using a fluorescent enzyme substrate resorufin β-D-galactopyranoside. Using this approach, the results demonstrated successful detection of 10 CFU mL^?1 of E. coli BL21 in simulated spinach wash water within 8?hours. Specificity of the assay was demonstrated with negative controls including Pseudomonas fluorescens and Listeria innocua.     

Effects of electrolyte concentration on the rotational dynamics of resorufin

We report on the rotational diffusion dynamics of the anionic chromophore resorufin in water and N-octyl-2-pyrrolidone (NOP) solutions as a function of solution electrolyte concentration. Our data show that resorufin exhibits a single exponential anisotropy decay in aqueous solutions containing up to 0.1 M lithium perchlorate (LiClO(4)). In contrast to the observed behavior of resorufin in pure NOP, where biexponential decay occurs, we also observe a single exponential anisotropy decay for resorufin in NOP with the addition of up to 0.1 M tetrabutylammonium bromide (TBAB) or tetraoctylammonium bromide (TOAB). For resorufin in NOP, the reorientation time constant increases with increasing electrolyte concentration, consistent with complexation between the resorufin anion and the electrolyte ammonium cation. We observe a qualitatively different trend in the aqueous resorufin solutions and understand these data for both solvent systems in the context of interactions between the chromophore and cationic species present.     

Chromogenic and fluorogenic signaling of sulfite by selective deprotection of resorufin levulinate

A new sulfite-selective probe system based on resorufin was investigated. Levulinate of resorufin exhibited a prominent chromogenic and turn-on type fluorogenic signaling toward sulfite ions in aqueous media based on the selective deprotection of the levulinate group. The sulfite-selective signaling was possible in the presence of commonly encountered anions.     

Activity of AC electrokinetically immobilized horseradish peroxidase

Dielectrophoresis (DEP) is an AC electrokinetic effect mainly used to manipulate cells. Smaller particles, like virions, antibodies, enzymes, and even dye molecules can be immobilized by DEP as well. In principle, it was shown that enzymes are active after immobilization by DEP, but no quantification of the retained activity was reported so far. In this study, the activity of the enzyme horseradish peroxidase (HRP) is quantified after immobilization by DEP. For this, HRP is immobilized on regular arrays of titanium nitride ring electrodes of 500 nm diameter and 20 nm widths. The activity of HRP on the electrode chip is measured with a limit of detection of 60 fg HRP by observing the enzymatic turnover of Amplex Red and H₂O₂ to fluorescent resorufin by fluorescence microscopy. The initial activity of the permanently immobilized HRP equals up to 45% of the activity that can be expected for an ideal monolayer of HRP molecules on all electrodes of the array. Localization of the immobilizate on the electrodes is accomplished by staining with the fluorescent product of the enzyme reaction. The high residual activity of enzymes after AC field induced immobilization shows the method's suitability for biosensing and research applications.     

Hemin/G-quadruplexes as DNAzymes for the fluorescent detection of DNA, aptamer-thrombin complexes, and probing the activity of glucose oxidase

Hemin/G-quadruplex catalyzes the H(2)O(2)-mediated oxidation of Amplex Red to the fluorescent product resorufin. This process is implemented to develop hairpin nucleic acid structures for the detection of DNA, to probe the catalytic activity of glucose oxidase, to use the thrombin-aptamer complex as a catalytic readout structure, and to quantitatively analyze telomere chain composition.     

Hydrogen peroxide-induced deacetylation of acetyl resorufin as a novel indicator reaction for fluorometric detection of glucose using only glucose oxidase

Hydrogen peroxide (H2O2)-induced deacetylation of non-fluorescent acetyl resorufin (1) to fluorescent resorufin (2) as a novel indicator reaction for fluorometric detection of glucose using only glucose oxidase (GOD) is described. When a 1:1:1 mixture of 1 (in CH3CN), glucose, and GOD (each in pH 7.4 phosphate buffer) was incubated at 25 degrees C under aerobic conditions, the resulting solution turned yellow to fluorescent pink due to 2. The formation of 2 was markedly retarded on incubation under anaerobic conditions. When a mixture of 1 and H2O2 was incubated under aerobic conditions, the formation of 2 was noted as in the case of the enzymatic reaction of 1. These results demonstrated that the observed color change is brought about through deacetylation of 1 to 2 induced by H2O2 generated in GOD-catalyzed oxidation of glucose. With regard to the fluorometric traces of the enzymatic reaction with 1 (0.2 mM), GOD (0.5 mg/ml), and glucose at 25 degrees C, fluorescence intensity exhibited a linear relationship against glucose concentration between 0.2 and 2.0 mm, with a correlation coefficient of 0.997. Neither ascorbic acid, uric acid, nor bilirubin significantly interfered with the transformation of 1 to 2 through GOD-catalyzed oxidation of glucose.     

Measurement of peroxidase activity in single neutrophils by combining catalyzed-enzyme reaction and epi-fluorescence microscopy

An epi-fluorescence microscopy for determination of peroxidase in individual neutrophils was developed by a combination of enzyme-catalyzed reaction and fluorescence detection. In this method, an individual cell was transferred into a microliter-volume vessel and lysed by freeze-thawing and ultrasonication. The peroxidases-catalyzed reaction was initiated by adding the buffer solution containing nonfluorescent substrates 10-acetyl-3,7-dihydroxyphenoxazine and H₂O₂ to the vessel. Peroxidase activity could be determined via measuring the fluorescent signal of the product resorufin of enzyme-catalyzed reaction. When the slope of kinetic curve of the enzyme-catalyzed reaction was used to quantify peroxidases in single cells, the effect of the time difference between each measurement and the interference from other intracellular compounds that could emit fluorescence can be eliminated.     

Research on the chemistry of phenoxazines

Resorufin (7-hydroxy-3-phenoxazinone) reacts with thiophenols to give 2,8-di(arylthio) derivatives. The site of entry of the nucleophilic residues indicates protonation of resorufin at the ring nitrogen atom. The changes in the absorption spectra of the arylthio derivatives of resorufin in the visible region as a function of the pH of the solution are associated with the acid-base dissociation of these compounds with respect to the hydroxyl group. The ionization constants of some 2,8- and 2,4,6,8-tetrasubstituted resorufin derivatives were measured.See [1] for communication VI.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 3, pp. 348–353, March, 1974.     

Development of an on-line high performance liquid chromatography detection system for human cytochrome P450 1A2 inhibitors in extracts of natural products

An on-line HPLC screening method for detection of inhibitors of human cytochrome P450 1A2 in extracts was developed. HPLC separation of extracts is connected to a continuous methoxyresorufin-O-demethylation (MROD) assay in which recombinant human P450 1A2 converts methoxyresorufin to its fluorescent metabolite resorufin. The system was tested with three P450 1A2 inhibitors, for which minimum detectable amounts (MDA) ranging from 0.7 to 9.5 ng were obtained. Analysis of a kava kava and a basil extract showed that the on-line system is applicable to complex mixtures, since in both extracts, peaks with P450 1A2 inhibiting activity were observed.     

The excited-state interaction of resazurin and resorufin with amines in aqueous solutions. Photophysics and photochemical reactions

The photophysics and photochemical behavior of the phenoxazin-3-one dyes, resazurin and resorufin, have been studied in aqueous solutions. The irradiation of resazurin in the presence of amines leads to deoxygenation of the N-oxide group, giving resorufin. This photoreaction is highly dependent on the amine structure and is efficient only in the presence of tertiary aliphatic amines. The absorption and fluorescence properties of these dyes are dependent on pH. At pH above 7.5 both dyes are in their anionic form. For resorufin this form is highly fluorescent (phiF = 0.75). At lower pH the fluorescence is strongly reduced. The N-oxide dye presents a very weak fluorescence quantum yield (0.11), which also is reduced at low pH. Flash photolysis experiments allowed characterization of the triplet state and the transients formed after irradiation of these dyes in the absence and presence of amines. The triplet quantum yields are 0.08 for resazurin and 0.04 for resorufin. The photodeoxygenation of N-oxide in the presence of amines occurs from the triplet state.     

A novel resorufin based fluorescent “turn-on” probe for the selective detection of hydrazine and application in living cells

In this study, a resorufin derivative RTP-1, which is a novel fluorescent ‘‘turn-on' probe for sensitive detection of hydrazine within 30 min, is designed and synthesized. The selective deprotection of the ester group of the probe by hydrazine led to a prominent enhancement of fluorescent intensity, as well as a remarkable color change from colorless to pink, which could be distinguished by naked eye. The fluorescence enhancement showed decent linear relationship with hydrazine concentration ranging from 0 to 50 mmol/L, with a detection limit of 0.84 mmol/L. The specificity of RTP-1 for hydrazine to a number of metal ions, anions and amines is satisfactory. The sensing mechanism of RTP-1 and hydrazine was evaluated by HPLC, ESI mass spectrometry and density functional theory(DFT).Moreover, we have utilized this fluorescent probe for imaging hydrazine in living cells, and the fluorescence was clearly observed when the cells were incubated with hydrazine(100 mmol/L) for 30 min.     

Synthesis of 7-Alkoxyquinolines,Coumarins, and Resorufins

Synthesis of the title compounds by treatment of the sodium salts of 7-quinolinol, 7-hydroxycoumarin, and resorufin with alkyl halides is described.