scholarly journals A single-molecule digital enzyme assay using alkaline phosphatase with a cumarin-based fluorogenic substrate

The Analyst ◽  
2015 ◽  
Vol 140 (15) ◽  
pp. 5065-5073 ◽  
Author(s):  
Yusuke Obayashi ◽  
Ryota Iino ◽  
Hiroyuki Noji
Keyword(s):  
Alkaline Phosphatase ◽  
Single Molecule ◽  
Enzyme Assay ◽  
Fluorogenic Substrate ◽  
Enzymatic Assays ◽  
Array Technology

Digitalization of fluorogenic enzymatic assays through the use of femtoliter chamber array technology is an emerging approach to realizing highly quantitative bioassays with single-molecule sensitivity.

Escherichia coli β-galactosidase is heterogeneous with respect to the activity of individual molecules

1998 ◽  
Vol 76 (5) ◽  
pp. 623-626 ◽  
Author(s):  
Douglas B Craig ◽  
Norman J Dovichi
Keyword(s):  
Escherichia Coli ◽  
Capillary Electrophoresis ◽  
Single Molecule ◽  
Enzyme Assay ◽  
Laser Induced Fluorescence ◽  
Enzyme Molecule ◽  
Fluorogenic Substrate ◽  
Individual Enzyme

Escherichia coli β-galactosidase molecules were incubated with fluorogenic substrate in a capillary. Upon flushing the reactor contents past the detector, individual randomly distributed peaks of product were observed, each representing the activity of an individual enzyme molecule. Individual molecules were found to differ with respect to their activities. Molecules showed a 23-fold distribution of activities with the majority of molecules within a 4-fold distribution.Key words: single-molecule chemistry, β-galactosidase, capillary electrophoresis, laser-induced fluorescence, enzyme assay.

scholarly journals Alkaline phosphatase interference in immuno-enzymatic assays.

2021 ◽  
Author(s):  
OSMAN Oğuz ◽  
Huriye Serin ◽  
Fatma Hocaoğlu Emre
Keyword(s):  
Alkaline Phosphatase ◽  
Liver Diseases ◽  
Troponin I ◽  
Total Error ◽  
Friedman Test ◽  
Acceptable Error ◽  
Enzymatic Assays ◽  
Serum Pool ◽  
Paired Samples ◽  
Immunoenzymatic Assay

Background: Alkaline phosphatase (ALP) enzymes are widely used as signal amplifiers in immunoenzymatic methods. Conditions that cause ALP elevations, such as bone or liver diseases can cause interference in immunoenzymatic methods. Objective: We aimed to examine ALP's effect on immunoenzymatic assay by adding isolated pure ALP to the prepared serum pool. Material and Methods: We prepared a serum pool and divided into 4 groups. By adding isolated pure ALP at different concentrations to each group, we obtained sample groups containing ALP enzyme at concentrations of 85 U/L, 340 U/L, 870 U/L and 1570 U/L. In each group, 20-repetition of βhCG, Ferritin, FT4, TSH, Troponin I and Vit B12 tests were performed. Coefficient of variation, bias, and total error were calculated. All groups were compared by using Friedman test for paired samples. Result: After ALP addition, the calculated total error values of FT4, βhCG and troponin I tests were found to be above the acceptable error limits. There were statistically significant differences in βhCG ,FT4, troponin I and Vit B12 tests when compared to the baseline ALP level (P<0,0125).Conclusion: Isolated ALP elevations can be a source of interference for immunoenzymatic methods.KeywordsAlkaline phosphatase, ALP, bias, immunoenzymatic, total error

scholarly journals Single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by FCS experiments

2020 ◽  
Vol 117 (35) ◽  
pp. 21328-21335
Author(s):  
Zhijie Chen ◽  
Alan Shaw ◽  
Hugh Wilson ◽  
Maxime Woringer ◽  
Xavier Darzacq ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Single Molecule ◽  
Particle Tracking ◽  
Single Particle ◽  
Theoretical Models ◽  
Single Particle Tracking ◽  
Correlation Spectroscopy ◽  
Single Molecule Level ◽  
Diffusion Phenomena ◽  
Diffusion Enhancement

Theoretical and experimental observations that catalysis enhances the diffusion of enzymes have generated exciting implications about nanoscale energy flow, molecular chemotaxis, and self-powered nanomachines. However, contradictory claims on the origin, magnitude, and consequence of this phenomenon continue to arise. To date, experimental observations of catalysis-enhanced enzyme diffusion have relied almost exclusively on fluorescence correlation spectroscopy (FCS), a technique that provides only indirect, ensemble-averaged measurements of diffusion behavior. Here, using an anti-Brownian electrokinetic (ABEL) trap and in-solution single-particle tracking, we show that catalysis does not increase the diffusion of alkaline phosphatase (ALP) at the single-molecule level, in sharp contrast to the ∼20% enhancement seen in parallel FCS experiments usingp-nitrophenyl phosphate (pNPP) as substrate. Combining comprehensive FCS controls, ABEL trap, surface-based single-molecule fluorescence, and Monte Carlo simulations, we establish thatpNPP-induced dye blinking at the ∼10-ms timescale is responsible for the apparent diffusion enhancement seen in FCS. Our observations urge a crucial revisit of various experimental findings and theoretical models––including those of our own––in the field, and indicate that in-solution single-particle tracking and ABEL trap are more reliable means to investigate diffusion phenomena at the nanoscale.

Sensitive fluorometry of heat-stable alkaline phosphatase (Regan enzyme) activity in serum from smokers and nonsmokers.

1983 ◽  
Vol 29 (2) ◽  
pp. 260-263 ◽  
Author(s):  
W C Maslow ◽  
H A Muensch ◽  
F Azama ◽  
A S Schneider
Keyword(s):  
Alkaline Phosphatase ◽  
Normal Limit ◽  
Heat Stability ◽  
Cross Reactivity ◽  
Fluorogenic Substrate ◽  
Normal Range ◽  
Heat Stable ◽  
Normal Individuals ◽  
Naphthoic Acid ◽  
The Mean

Abstract We developed a simple, sensitive enzymatic assay involving the fluorogenic substrate naphthol AS-MX phosphate [(3-hydroxy-2-naphthoic acid 2,4-dimethylanilide) phosphate] to measure heat-stable alkaline phosphatase (EC 3.1.3.1), the Regan isoenzyme, in human serum. The day-to-day CV was 5.7% for a serum activity of 0.080 arbitrary units/L. Measurable amounts of enzyme were detected in most normal individuals. The mean for 51 nonsmokers was 0.068 (SD 0.037) arb. units/L; for 25 smokers it was 0.440 (SD 0.360) arb. units/L. Activity of this isoenzyme in smokers was as much as 10-fold the upper normal limit for nonsmokers. Activation of this tumor marker by smoking has not received attention hitherto. We conclude that a truly normal range can only be established among nonsmokers. The isoenzymes in smokers, nonsmokers, and pregnant women were similar in their heat stability, immunologic cross reactivity, and inhibition by L-phenylalanine.

scholarly journals Plasma extracellular vesicles detected by Single Molecule array technology as a liquid biopsy for colorectal cancer

2020 ◽  
Vol 9 (1) ◽  
pp. 1809765 ◽  
Author(s):  
Ping Wei ◽  
Fei Wu ◽  
Bin Kang ◽  
Xiaohua Sun ◽  
Fabienne Heskia ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Extracellular Vesicles ◽  
Single Molecule ◽  
Liquid Biopsy ◽  
Array Technology

Measurement of Bone-Specific Alkaline Phosphatase by an Immunoselective Enzyme Assay Method

1996 ◽  
Vol 33 (2) ◽  
pp. 127-131 ◽  
Author(s):  
Keishi Hata ◽  
Hisako Tokuhiro ◽  
Kiyoshi Nakatsuka ◽  
Takami Miki ◽  
Yoshiki Nishizawà ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Enzyme Assay ◽  
Metabolic Diseases ◽  
Clinical Investigation ◽  
Assay Method ◽  
Osteosarcoma Cell ◽  
Electrophoretic Method ◽  
Specific Alkaline Phosphatase ◽  
Bone Specific Alkaline Phosphatase ◽  
Age Related

We evaluated a new immunoselective enzyme assay of bone-specific alkaline phosphatase (ALP). The monoclonal antibody used in this assay was raised against purified bone-specific ALP obtained from SAOS-2 human osteosarcoma cell line. Calibration was based on the enzyme's own activity. The relative activity of the antibody was 100% with bone ALP, 8·7% with liver ALP, and 0% with placental and intestinal ALPs. Intra- and inter-assay coefficients of variation were less than 4%. The sensitivity of the assay was 0·7 U/L, and the linearity extended from 2 to 140 U/L. The recovery of bone-specific ALP standard added to serum was 94–106%. The correlation coefficient between this method and the polyacrylamide gel (PAG) electrophoretic method was 0·94. The mean value of bone-specific ALP in 89 healthy adults (mean age 29 years, SD 5 years) was 18·5 U/L (SD 4·1 U/L). Interestingly, mean bone-specific ALP activities in 60 premenopausal women (mean age 39 years, SD 8 years) and 70 postmenopausal women (mean age 57 years, SD 5 years) were 20·3 U/L (SD 6·5 U/L) and 31·1 U/L (SD 11·1 U/L), respectively. The age-related increase in bone-specific ALP was significant and more pronounced in women ( P < 0·01). We conclude that this new immunoassay of bone-specific ALP would be useful for clinical investigation of patients with osteoporosis or other metabolic diseases of bone.

Reliable Digital Single Molecule Electrochemistry for Ultrasensitive Alkaline Phosphatase Detection

2016 ◽  
Vol 88 (18) ◽  
pp. 9166-9172 ◽  
Author(s):  
Zhen Wu ◽  
Chuan-Hua Zhou ◽  
Liang-Jun Pan ◽  
Tao Zeng ◽  
Lian Zhu ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Single Molecule
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