Detection Of Alkaline Phosphatase Research Articles

Excimer-based Activatable Fluorescent Sensor for Sensitive Detection of Alkaline Phosphatase

Excimer-based Activatable Fluorescent Sensor for Sensitive Detection of Alkaline Phosphatase

Facile preparation of red emission Hg-doped ZnSe QDs and ratiometric determination of alkaline phosphatase based on in situ generation of dual fluorescent reporter

In this study, highly red emissive mercury-doped zinc selenide quantum dots (Hg-doped ZnSe QDs) were prepared by facile mixing of Hg2+ and ZnSe QDs. Further, a ratio fluorometric technique was designed for ultrasensitive detection of alkaline phosphatase (ALP) based on in situ generation of Hg-doped ZnSe QDs and quinoxaline derivative (QXD). Under the catalysis of ALP, ascorbic acid (AA) was generated from ascorbic acid 2-phosphate, and the as-generated AA underwent a redox reaction with Hg2+ to produce dehydroascorbic acid (DHA) and Hg, which resulted in significant suppression of red fluorescence at 635 nm because the lack of Hg2+ blocked the formation of Hg-doped ZnSe QDs. Furthermore, the occurrence of reaction between DHA and o-phenylenediamine produced fluorescent QXD, which caused the significant enhancement of blue fluorescence at 436 nm. Thus, a novel ratio nanoprobe utilizing Hg-doped ZnSe QDs and QXD as dual fluorescent reporter was fabricated for measuring ALP activity. The linear ranges were 0.001–0.02 U L−1 and 0.02–1.4 U L−1 with the detection limit of 0.001 U L−1. Furthermore, the nanoprobe enabled visual detection of ALP activity when exposed to ultraviolet light, and it exhibited satisfactory results for measuring ALP activity in human serum. This study provides a valuable method for ALP-related disease diagnosis and drug screening.

Polydopamine-assisted immobilization of silk fibroin and its derived peptide on chemically oxidized titanium to enhance biological activity in vitro

Biochemical modification can endow the surface of implants with superior biological activity. Herein, silk fibroin (SF) protein and its anionic derivative peptides (Cs) were covalently immobilized onto a titanium implant surface via a polydopamine layer. The successful conjugation of SF and Cs was revealed by X-ray photoelectron spectroscopy (XPS), field-emission scanning electron microscopy (FE-SEM), atomic force microscopy (AFM), and contact angle measurements. The addition of Cs prevented the conformational transition of silk fibroin to silk II. The deposition of apatite on its surface was significantly accelerated, and the bioactive composite coating was observed to enhance protein adsorption and cell proliferation. More importantly, it also promoted the osteogenic differentiation of bone marrow stem cells (BMSCs) for the quantitative and qualitative detection of alkaline phosphatase (ALP) and alizarin red (ARS). Overall, the stable performance and enhanced osteogenic property of the composite coating promote an extensive application for clinical titanium-based implants.

CDs–MnO2–TPPS Ternary System for Ratiometric Fluorescence Detection of Ascorbic Acid and Alkaline Phosphatase

Manganese dioxide (MnO2) nanosheet-based fluorescence sensors often use oxidase-like activity or wide absorption spectrum for detection of antioxidants. In those strategies, MnO2 nanosheets were reduced to Mn2+ by antioxidants. However, few strategies emphasize the role of Mn2+ obtained from MnO2 reduction in the design of the fluorescence sensor. Herein, we expanded the application of a MnO2 nanosheet-based fluorescence sensor by involving Mn2+ in the detection process using ascorbic acid (AA) as a model target. In this strategy, carbon dots (CDs), MnO2 nanosheets, and tetraphenylporphyrin tetrasulfonic acid (TPPS) comprise a ternary system for ratiometric fluorescence detection of AA. Initially, CDs were quenched by MnO2 nanosheets based on the inner filter effect, while TPPS maintained its fluorescence intensity. After the addition of AA, MnO2 nanosheets were reduced to Mn2+ so that the fluorescence intensity of CDs was recovered and TTPS was quenched by coordination with Mn2+. Overall, AA triggered an emission intensity increase at 440 nm for CDs and a decrease at 640 nm for TPPS. The ratio intensity of CDs to TPPS (F440/F640) showed a good linear relationship from 0.5 to 40 μM, with a low detection limit of 0.13 μM for AA detection. By means of the alkaline phosphatase (ALP)-triggered generation of AA, this strategy can be applied for the detection of ALP in the range of 0.1–100 mU/mL, with a detection limit of 0.04 mU/mL. Furthermore, this sensor was applied to detect AA and ALP in real, complex samples with ideal recovery. This novel platform extended the application of MnO2 nanosheet-based fluorescence sensors.

Temporal induction of Lhx8 by optogenetic control system for efficient bone regeneration

BackgroundThe spatiotemporal regulation of essential genes is crucial for controlling the growth and differentiation of cells in a precise manner during regeneration. Recently, optogenetics was considered as a potent technology for sophisticated regulation of target genes, which might be a promising tool for regenerative medicine. In this study, we used an optogenetic control system to precisely regulate the expression of Lhx8 to promote efficient bone regeneration.MethodsQuantitative real-time PCR and western blotting were used to detect the expression of Lhx8 and osteogenic marker genes. Alkaline phosphatase staining and alizarin red staining were used to detect alkaline phosphatase activity and calcium nodules. A customized optogenetic expression system was constructed to regulate Lhx8, of which the expression was activated in blue light but not in dark. We also used a critical calvarial defect model for the analysis of bone regeneration in vivo. Moreover, micro-computed tomography (micro-CT), three-dimensional reconstruction, quantitative bone measurement, and histological and immunohistochemistry analysis were performed to investigate the formation of new bone in vivo.ResultsDuring the osteogenic differentiation of BMSCs, the expression levels of Lhx8 increased initially but then decreased thereafter. Lhx8 promoted the early proliferation of BMSCs but inhibited subsequent osteogenic differentiation. The optogenetic activation of Lhx8 in BMSCs in the early stages of differentiation by blue light stimulation led to a significant increase in cell proliferation, thus allowing a sufficient number of differentiating BMSCs to enter the later osteogenic differentiation stage. Analysis of the critical calvarial defect model revealed that the pulsed optogenetic activation of Lhx8 in transplanted BMSCs over a 5-day period led to a significant increase in the generation of bone in vivo.ConclusionsLhx8 plays a critical role in balancing proliferation and osteogenic differentiation in BMSCs. The optogenetic activation of Lhx8 expression at early stage of BMSCs differentiation led to better osteogenesis, which would be a promising strategy for precise bone regeneration.

Restoring the Oxidase-Like Activity of His@AuNCs for the Determination of Alkaline Phosphatase.

In this paper, we propose a simple colorimetric method for the sensitive and selective detection of alkaline phosphatase (ALP) activity based on the turn off/turn on oxidase mimic activity of His@AuNCs. His@AuNCs/graphene oxide hybrids (His@AuNCs/GO) were easily obtained using the self-assembly method with poly (diallyldimethylammonium chloride) (PDDA)-coated GO and showed high oxidase-like activity compared with His@AuNCs. We found that the pyrophosphate ion (P2O74−, PPi) could effectively inhibit the oxidase mimic activity of His@AuNCs/GO, and the hydrolysis of PPi by ALP restored the inhibited activity of His@AuNCs/GO, enabling them to efficiently catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to generate the blue oxidized product oxTMB. The intensity of the color showed a linear dependency with the ALP activity. ALP was detected in the linear range of 0–40 mU/mL with a low detection limit (LOD) of 0.26 mU/mL (S/N = 3). The proposed method is fast, easy, and can be applied to monitor the ALP activity in serum samples accurately and effectively, which suggests its practicability and reliability in the detection of ALP activity in clinical practice.

Size-dependent light scattering of CoOOH nanoflakes for convenient and sensitive detection of alkaline phosphatase in human serum.

As a natural enzyme, alkaline phosphatase (ALP) plays an essential role in clinicopathological examinations and biomedical research, and is capable of hydrolyzing the phosphate group of l-ascorbic acid-2-phosphate (AAP) to yield l-ascorbic acid (L-AA). L-AA reduced cobalt oxyhydroxide (CoOOH) nanoflakes to Co2+ , leading to a smaller size and weaker light scattering, which could be monitored by electron microscopic images and optical spectra. The indirect detection of ALP was achieved by the reduced light scattering signal of CoOOH nanoflakes. Under optimal conditions, the decrease in scattering intensity was proportional to the ALP concentration over the range 0.1-160 U/L and the detection limit was 0.034 U/L (3σ/k). Compared with other assays, this proposed light scattering method was more convenient and economic for ALP sensing. The method was successfully applied to ALP analysis in human serum samples, and was similar to the results obtained by commercial kits.

Dual-readout test strips platform for portable and highly sensitive detection of alkaline phosphatase in human serum samples

In this work, a very simple dual-readout lateral flow test strip (LFTS) platform was developed for sensitive detection of alkaline phosphatase (ALP) based on a portable device. In this assay, quantum dots (QDs) conjugated with bovine serum albumin (QDs-BSA) were chosen as fluorescence signal labels. In the absence of ALP, MnO2 nanosheets aggregate on the test line and exhibit an obvious brown color, which can be observed by naked eyes to realize semi-qualitative analysis. Meanwhile, fluorescence intensity of QDs-BSA can also be effectively quenched by MnO2 nanosheets due to inner-filter effect. Correspondingly, in the presence of ALP, ALP can catalyze the hydrolysis of ascorbic acid 2-phosphate (AAP) to generate L-ascorbic acid (AA), which can reduce MnO2 into Mn2+, accompanying with the obvious fluorescence recovery of the QDs. By simply monitoring the change of colorimetric and fluorescent signal on the test line, trace amount of ALP can be quantitatively detected. Under the optimal conditions, measurable evaluation of ALP was reached in a linear range from 1 U/L to 20 U/L with a detection limit of 0.7 U/L based on fluorescence signal. Furthermore, this colorimetric/fluorescent dual-readout assay was successfully applied to monitor ALP in human serum samples, showing its great potential as a point of care biosensor for clinical diagnosis.

Novel ratiometric fluorescent probe for real-time detection of alkaline phosphatase and its application in living cells

A novel ratiometric fluorescent probe has been developed through a simple synthetic route for the detection of alkaline phosphatase(ALP) in aqueous media and for fluorescence imaging in living cells. The introduction of a spontaneous-degradation spacer in the design of the fluorescent probe is beneficial for the ratio detection method and allows the selection of a fluorophore with an amino group. Under catalysis by ALP, the phosphate monoester bond breaks; this is followed by 1,4-elimination, decomposition of the carbamate moiety, and subsequent formation of the 4-amine-1,8-naphthalimide fluorophore. The probe APN shows a significant fluorescence colour change from blue to green in response to ALP, and the fluorescence intensity ratio of the probe solution (F550/F480) has a good linear relationship with the ALP concentration in the range of 0 to 100 U L-1. Our studies have demonstrated that APN exhibits high accuracy in recognising ALP, with a detection limit as low as 0.16 U L-1. Furthermore, the probe shows very good biocompatibility, which is beneficial for its application in biological systems.

MnO2 shell-isolated SERS nanoprobe for the quantitative detection of ALP activity in trace serum: Relying on the enzyme-triggered etching of MnO2 shell to regulate the signal

• A MnO 2 shell-isolated SERS nanoprobe (Au-Mpy-Au-MnO 2 ) is prepared for quantitative detection of ALP activity. • Due to the efficient physical isolation of MnO 2 shell, the nanoprobe demonstrates high stability against salt and thiol. • The sensing performance is achieved by enzyme-triggered etching of MnO 2 shell. • With MnO 2 shell as Raman reference, this system effectively eliminates the experimental interferences. • Integrating with in-capillary technique, this SERS system is capable of direct quantitative detection in trace serum. Establishing a stable, reliable and sensitive assay for the direct analysis in trace serum is of significance for the medical diagnosis and disease monitoring. We develop a MnO 2 shell-isolated SERS nanoprobe (Au-Mpy-Au-MnO 2 , AMAM) for the quantitative detection of alkaline phosphatase (ALP) in trace serum. Due to the physical isolation of MnO 2 shell, AMAM is equipped with high stability, rarely happening the accidental aggregation. Particularly, the SERS signal is regulated by the etching of MnO 2 shell, requiring not to change the local electromagnetic field or the molecular structure of Raman reporter (4-mercaptopyridine (Mpy)). Designating MnO 2 shell as the Raman reference and the ratio of I Mpy /I MnO2 as the response signal, AMAM system can obviously eliminate the experimental interference and achieve highly reliable SERS quantification for ALP. This ratiometric SERS system exhibits good reproducibility, high stability and sensitivity for the ALP analysis. AMAM system is able to perform the colorimetric and SERS dual-readout detection, which displays a wide linear dynamic range (0.1–70 U/L) for ALP with a detection limit of 0.079 U/L. Additionally, integrating with in-capillary SERS technique, our proposed nanoprobe is also applicable of direct SERS analysis in trace undiluted human serum (2 μL).

A novel electronic assay based on a sol-gel transition reaction and a thin-film transistor of supramolecular hydrogels to detect alkaline phosphatase activity

Abstract Herein, we developed an extended floating-gate thin-film transistor (EFG-TFT) together with various sol-gel phases to determine alkaline phosphatase (ALP) activity. The EFG-TFT was fabricated with ZnO as the channel material and YOX as the dielectric layer, which provided an extended area for sensing the analyte interacting with the designed hydrogelator. A peptide-based supramolecular hydrogel material, i.e., 2-naphthylacetic acid- l -phenylalanine- l -phenylalanine- l -O-phosphor tyrosine (named Nap-FF-Yp) was synthesized as our sensing platform to detect ALP activity and the sol-gel state. Nap-FF-Yp could be cleaved by ALP during hydrolysis, leading to π-π stacking, and finally changed to a hydrogel. Due to the difference in the potential drop between the liquid phase and gel phase of the hydrogel, the sol-gel transition reaction could be monitored by the electrical signal. The greater the extent of hydrogelation, the smaller the threshold voltage (Vth) variation, and the threshold voltage shifted to the left. Under the device’s optimal conditions with oxygen plasma treatment, the limit of detection (LOD) reached 0.024 U/L. The development of this novel sol-gel state for a high-speed screening sensing platform with an excellent electrical response demonstrates the potential capabilities of future high-speed response sensors.

An Exonuclease I-Aided Turn-Off Fluorescent Strategy for Alkaline Phosphatase Assay Based on Terminal Protection and Copper Nanoparticles.

As an important DNA 3′-phosphatase, alkaline phosphatase can repair damaged DNA caused by replication and recombination. It is essential to measure the level of alkaline phosphatase to indicate some potential diseases, such as cancer, related to alkaline phosphatase. Here, we designed a simple and fast method to detect alkaline phosphatase quantitively. When alkaline phosphatase is present, the resulting poly T-DNA with a 3′-hydroxyl end was cleaved by exonuclease I, prohibiting the formation of fluorescent copper nanoparticles. However, the fluorescent copper nanoparticles can be monitored with the absence of alkaline phosphatase. Hence, we can detect alkaline phosphatase with this turn-off strategy. The proposed method is able to quantify the concentration of alkaline phosphatase with the LOD of 0.0098 U/L. Furthermore, we utilized this method to measure the effects of inhibitor Na3VO4 on alkaline phosphatase. In addition, it was successfully applied to quantify the level of alkaline phosphatase in human serum. The proposed strategy is sensitive, selective, cost effective, and timesaving, having a great potential to detect alkaline phosphatase quantitatively in clinical diagnosis.

A fluorometric and optical signal dual-readout detection of alkaline phosphatase activity in living cells based on ATP-mediated porphyrin MOFs

Alkaline phosphatase (ALP), as an important biomarker, is closely associated with various diseases. Therefore, detecting the ALP activity is of considerable importance to diagnosis and treatment of diseases. In this study, we have first exploited porphyrin-adenosine triphosphate-based nanoscale chiral metal-organic framework (denoted as NPCN-222-ATP) for the quantitative determination the activity of ALP through fluorometric and optical signal dual-readout mode. In this detection system, the ALP-catalyzed hydrolysis of chiral ATP molecules leads to the recovery of the fluorescence of porphyrin, accompanied by the gradual decrease of the circular dichroism (CD) signal. This probe showed excellent selectivity and sensitivity for ALP activity with the limits of detection of 0.0240 mU/mL and 0.0225 mU/mL for the fluorometric and optical signal readout mode, respectively, which were significantly better than the current ALP detection system. More importantly, such chiral probe successfully realized the monitoring and imaging of ATP activity in living cells, providing a new way for chiral MOFs to be used in biological analysis and detection platform.

Target-induced transcription amplification to trigger the trans-cleavage activity of CRISPR/Cas13a (TITAC-Cas) for detection of alkaline phosphatase

Herein, an ultra-sensitive alkaline phosphatase (ALP) sensing strategy is developed by target-induced transcription amplification to trigger the trans-cleavage activity of Cas13a (TITAC-Cas). A double-stranded DNA duplex integrating a T7 promoter with 5′-phosphate and a transcription template (5′P-dsDNA) serves as the ALP substrate. In the absence of ALP, 5′P-dsDNA can be degraded by the λexo, leading to the subsequent transcription failure. In the presence of ALP, dephosphorylation reaction converts the 5′P-dsDNA to 5′OH-dsDNA and provides the protection for T7 promoter against the λexo-digestion. The intact T7 promoter of 5′OH-dsDNA can activate T7 transcription to produce a mass of single-stranded RNA (ssRNA). The ssRNA products possess a full complementarity to the spacer of crRNA and activate the ssRNase activity of CRISPR/Cas13a. As a result, Cas13a exhibits the indiscriminate cleavage of collateral FQ-reporter to release significant fluorescence signal, realizing the ultra-sensitive detection of ALP. Due to the triple signal amplification (ALP self-catalysis, T7 transcription amplification, and trans-cleavage of CRISPR/Cas13a), TITAC-Cas assay shows the ultra-sensitive detection of ALP activity with a wide linear range from 0.008 to 250 U∙L−1). The LOD is calculated to be 6 ± 0.52 mU∙L−1. TITAC-Cas assay is also successfully applied for analysis of ALP activity in HepG2 cell lysate with high fidelity. In addition, this method is employed to screen ALP inhibitor.

Development of a new chromogenic medium for the enumeration of Bacillus cereus in various ready-to-eat foods

Enumeration of Bacillus cereus in ready-to-eat food with high background flora has been challenging due to the low selectivity and recoverability of the selective media. In this study, a new medium for detection of B. cereus was developed. This medium, B. cereus chromogenic agar (BCCA), contains an egg yolk emulsion for detection of lecithinase activity, a chromogenic substrate specific for detecting alkaline phosphatase activity, and three antibiotics for selectivity. The aim of this study was to compare the performance of BACCA with that of two conventional selective media (MYPA and PEMBA) and two commercialized chromogenic media (CBC agar and Bacara medium). The newly developed media has been evaluated for its inclusivity/exclusivity and recoverability in pure culture and in artificially inoculated ready-to-eat foods, respectively. BCCA exhibited similar or better recoverability and inclusivity/exclusivity for enumeration of B. cereus and B. thuringiensis from pure cultures than two conventional [mannitol yolk polymyxin B agar (MYPA) and polymyxin pyruvate egg yolk mannitol bromothymol blue agar (PEMBA)] and two commercial chromogenic media [chromogenic B. cereus (CBC) agar and Bacara medium]. Furthermore, BCCA yielded similar or enhanced levels of recoverability and superior selectivity of B. cereus and B. thuringiensis from ready-to-eat food products, particularly radish sprouts and a sprout mix, compared with the other media tested. These findings indicate that BCCA comprises a useful option for the isolation and enumeration of B. cereus from commercial foods.

MicroRNA-214-3p Suppresses Ankylosing Spondylitis Fibroblast Osteogenesis via BMP–TGFβ Axis and BMP2

Recent investigations suggest microRNAs (miRs) exert functions in fibroblast osteogenesis in ankylosing spondylitis (AS), an inflammatory rheumatic disease. But the mechanism of miR-214-3p in osteogenic differentiation in AS is not clearly understood yet. In this study, fibroblasts were obtained from the capsular ligament of patients with AS and femoral neck fracture and cultured for osteogenic induction and identified. The roles of miR-214-3p and bone morphogenic protein 2 (BMP2) in AS fibroblast osteogenesis were assessed via gain- and loss-of-function, alizarin red S staining, and alkaline phosphatase (ALP) detection. Levels of miR-214-3p, BMP2, osteogenic differentiation-related proteins, and BMP–TGFβ axis-related proteins were further measured. Consequently, miR-214-3p was downregulated in AS fibroblasts, with enhanced ALP activity and calcium nodules, which were reversed by miR-214-3p overexpression. BMP2 was a target gene of miR-214-3p and promoted AS fibroblast osteogenesis by activating BMP–TGFβ axis, while miR-214-3p inhibited AS fibroblast osteogenesis by targeting BMP2. Together, miR-214-3p could prevent AS fibroblast osteogenic differentiation by targeting BMP2 and blocking BMP–TGFβ axis. This study may offer a novel insight for AS treatment.

Substrate-Independent Laser-Induced Graphene Electrodes for Microfluidic Electroanalytical Systems

Laser-induced graphene’s (LIG) inherent graphene-like and highly porous characteristics and its simple, scalable, and inexpensive fabrication render it a desirable electrode material for bio- and chemosensors. The best LIG electrodes are made in polyimide foils using a CO2 laser scriber, which unfortunately limits their integration into more sophisticated analytical devices due to polyimide’s inertness. The transfer of LIG electrodes onto standard polymer substrates used in microfluidic systems and their use in microfluidic assays were therefore studied and the resulting electrodes characterized morphologically, chemically, and electroanalytically. It was found that a direct pressure-driven transfer produces highly functional transfer-LIG (tLIG) electrodes. tLIG differed from LIG electrodes with respect to a much smoother surface and hence a lower active surface area, a loss of the graphene characteristic Raman 2D peak, and a slight decrease in electron transfer rates. However, their performance in amperometric detection strategies were comparable also when used in adhesive-tape-enabled microfluidic channels for the detection of p-aminophenol. tLIG outperformed LIG electrodes in their ability to be integrated into more advanced microfluidic channel systems made of an all-polymethyl methacrylate (PMMA) substrate for the biosensing detection of alkaline phosphatase, commonly used as a biomarker and as a biosensor amplification system. LIG and tLIG have hence the potential to change electroanalytical sensing in diagnostic systems as their fabrication requires minimal resources, is highly scalable, and allows their integration into simple and, as tLIG, also sophisticated analytical systems.

Smart nanozyme of silver hexacyanoferrate with versatile bio-regulated activities for probing different targets

Smart nanozymes that can be facile and rapidly produced, while with efficiently bio-regulated activity, are attractive for biosensing applications. Herein, a smart nanozyme, silver hexacyanoferrate (Ag4[Fe(CN)6]), was constructed in situ via the rapid, direct reaction between silver(I) and K4[Fe(CN)6]. And the activity of the nanozyme can be rationally modulated by different enzymatic reactions including the glucose oxidase (GOx, taken as a model oxidoreductase), alkaline phosphatase (ALP), and acetylcholinesterase (AChE). On the basis of which, a multiple function platform for the highly sensitive detection of glucose, ALP and AChE were developed through colorimetry. Corresponding detection limits for the above three targets were found to be as low as 0.32 μM, 3.3 U/L and 0.083 U/L (S/N = 3), respectively. The present study provides a novel nanozyme that can be produced in situ, which rules out the harsh, cumbersome, and time-consuming synthesis/purification procedures. In addition, it establishes a multiple function platform for the amplified detection of versatile targets by the aid of the developed nanozyme, whose detection has the advantages of low cost, ease-of-use, high sensitivity, and good selectivity.

A ratiometric lanthanide fluorescent probe for highly sensitive detection of alkaline phosphatase and arsenate

A novel dual-ligand lanthanide coordination polymer nanoparticles (ThT@luminol-Eu-GMP CPNPs) were synthesized for detecting alkaline phosphatase (ALP) and arsenate. The ThT@luminol-Eu-GMP CPNPs were constructed through self-assemble of central ion (Eu3+) with bridging ligands of guanosine phosphate (GMP) and luminol, and simultaneously embedded the guest dye molecule thioflavin T (ThT) during the process. This design boosted the fluorescent performance of ThT through two paths. The molecular rotation of ThT was restricted due to the tight embedding of ThT in CPNPs, resulting in an increased fluorescence of ThT on one hand. On the other hand, the overlapped spectra between the emission spectra of luminol and the absorption spectra of ThT induced the FRET effect, which further facilitated the fluorescence of ThT. In the presence of ALP, the ingenious structure of ThT@luminol-Eu-GMP was destroyed due to the specific shear of ALP to GMP, which not only got rid of the limiting effect of ThT, but also broken the FRET between luminol and ThT, leading to a sharply decreased fluorescence of ThT. Meanwhile, the AIE effect of the luminol was promoted, leading to an increased fluorescence of luminol. According to ratio of the fluorescence of luminol to ThT, ALP can be monitored sensitively. Moreover, the ThT@luminol-Eu-GMP can also be used to sensitively detect arsenate based on the inhibition of arsenate for ALP.

Quantification of alkaline phosphatase in cell lysates by a simple fluorescence method in combination with a modified standard addition calibration strategy

Alkaline phosphatase (ALP) is an important diagnostic indicator of many human diseases. Its detection is of great importance in fields such as clinical diagnosis and biomedical research. In this contribution, a simple label-free fluorescence sensing method for ALP detection was designed by integrating ALP triggered hydrolysis, terminal deoxynucleotidyl transferase assisted DNA polymerization and complex formation between G-quadruplex and thioflavin T together. Furthermore, in order to mitigate the influence of matrix effects and background interference effects on the quantitative results of the proposed fluorescence sensing method encountered in the analysis of real-world complex biological samples, the probe technique-based generalized multivariate standard addition strategy (GMSAprobe) was specifically modified, and combined with the proposed fluorescence sensing method to achieve satisfactory quantitative results for ALP in cell lysate samples with accuracy comparable to that of a commercial ALP assay kit. Its limit of detection and limit of quantification values were about 0.02 and 0.07 U/L, respectively, more than 20 times lower than those of the commercial ALP assay kit. Due to its merits of simplicity, sensitivity, specificity, as well as robustness to matrix effects and background interference effects, the combination of the proposed fluorescence sensing method and the modified GMSAprobe has a great potential for ALP assay in biochemical research and clinical diagnosis.