Ascorbic Acid 2-phosphate Research Articles

Near-infrared light-induced photoelectrochemical biosensor based on plasmon-enhanced upconversion nanocomposites for microRNA-155 detection with cascade amplifications

Herein, a novel near-infrared (NIR) light-driven photoelectrochemical (PEC) biosensor based on NaYF4:Yb3+, Er3+@Bi2MoO6@Bi (NYF@BMO@Bi) nanocomposites was elaborately developed to achieve highly sensitive detection of microRNA-155 (miRNA-155). To realize signal enhancement, the coupled plasmonic bismuth (Bi) nanoparticles were constructed as an energy relay to facilitate the transfer of energy from NaYF4:Yb3+, Er3+ to Bi2MoO6, ultimately enabling the efficient separation of electron-hole pairs of Bi2MoO6 under the irradiation of a 980 nm laser. For constructing biosensing system, the initial signal was firstly amplified after the addition of alkaline phosphatase (ALP) in conjunction with the biofunctionalized NYF@BMO@Bi nanocomposites, which could catalyze the conversion of ascorbic acid 2-phosphate into ascorbic acid, and then consumed the photoacoustic holes created on the surface of Bi2MoO6 for the enlarging photocurrent production. Upon addition of target miRNA-155, the cascade signal amplification process was triggered while the ALP-modified DNA sequence was replaced and then followed by the initiation of a simulated biocatalytic precipitation reaction to attenuate the photocurrent response. On account of the NIR-light-driven and cascade amplifications strategy, the as-constructed biosensor was successfully utilized for the accurate determination of miRNA-155 ranging from 1 fM to 0.1 μM with a detection limit of 0.32 fM. We believed that the proposed nanocomposites-based NIR-triggered PEC biosensor could provide a promising platform for effective monitoring other tumor biomarkers in clinical diagnostics.

A Strategy for the Determination of Alkaline Phosphatase Based on the Self-Triggered Degradation of Metal-Organic Frameworks by Phosphate.

Alkaline phosphatase (ALP) is widely present in the human body and is an important biomarker. Numerous ALP detection studies have been carried out, and ascorbic acid (AA) is often used as the reducing component in the sensors to monitor ALP levels since it can be produced from ascorbic acid 2-phosphate (AA2P) hydrolysis in the presence of ALP. However, it is well-known that AA is a strong reducing agent and can be easily oxidized. The disproportion between oxidized AA and reduced AA reactions results in the generation of AA free radicals with single electrons that may lead to inaccurate results in assays. To solve this problem, we synthesized a core-shell metal-organic framework sensor (PATP-Au@ZIF-8 NP) and used it as a sensitive and accurate ALP detection sensor with self-triggered control of phosphate ions (Pi) to avoid the potential inaccuracy of the method that uses AA as the reducing component. By establishing a physical shell on the surface of the gold nanoparticles (Au NPs), the sensor not only can eliminate the random assembly of metal nanoparticles caused by plasma exposure but also can generate self-triggering of Pi caused by ALP. Pi can decompose ZIF-8 through coordination with Zn2+ and thus can destroy the ZIF-8 shell structure of the prepared PAZ NPs. Au NPs are released and then become aggregated, in turn causing the SERS "hot spot" area to increase. The enhancement of the SERS signals was found to be directly associated with the level of Pi released from ALP-triggered hydrolysis. The response of the strategy was linear at ALP concentrations ranging from 0.1 to 150 mU/mL (r = 0.996) with a detection limit of 0.03 mU/mL. Lastly, the developed strategy was employed in the evaluation of ALP inhibitors, and the possibility to implement the developed SERS strategy for rapid and selective analysis of ALP in human serum was demonstrated.

Culture of leukocyte-derived cells from human peripheral blood: Increased expression of pluripotent genes OCT4, NANOG, SOX2, self-renewal gene TERT and plasticity.

There are few stem cells in human peripheral blood (PB). Increasing the population and plasticity of stem cells in PB and applying it to regenerative medicine require suitable culture methods. In this study, leukocyte populations 250 mL of PB were collected using a blood separator before that were cultured in optimal cell culture medium for 4 to 7 days. After culturing, stemness characteristics were analyzed, and red blood cells were removed from the cultured cells. In our results, stemness markers of the leukocyte populations Sca-1+ CD45+, CD117+ CD45+, and very small embryonic-like stem cells CD34+ Lin- CD45- and CXCR4+ Lin- CD45- were significantly increased. Furthermore, the expression of stem cell genes OCT4 (POU5F1), NANOG, SOX2, and the self-renewal gene TERT was analyzed by quantitative real-time polymerase chain reaction in these cells, and it showed a significant increase. These cells could be candidates for multi-potential cells and were further induced using trans-differentiation culture methods. These cells showed multiple differentiation potentials for osteocytes, nerve cells, cardiomyocytes, and hepatocytes. These results indicate that appropriate culture methods can be applied to increase expression of pluripotent genes and plasticity. Leukocytes of human PB can be induced to trans-differentiate into pluripotent potential cells, which will be an important breakthrough in regenerative medicine.

Effect of glucose depletion and fructose administration during chondrogenic commitment in human bone marrow-derived stem cells

BackgroundBone marrow mesenchymal stromal cells (BMSCs) are promising for therapeutic use in cartilage repair, because of their capacity to differentiate into chondrocytes. Often, in vitro differentiation protocols employ the use of high amount of glucose, which does not reflect cartilage physiology. For this reason, we investigated how different concentrations of glucose can affect the chondrogenic differentiation of BMSCs in cell culture pellets. Additionally, we investigated how fructose could influence the chondrogenic differentiation in vitro.MethodsBMSC were isolated from six donors and cultured in DMEM containing glucose at either 25 mM (HG), 5.5 mM (LG) or 1 mM (LLG), and 1% non-essential amino acids, 1% ITS+, in the presence of 100 nM dexamethasone, 50 µg/ml ascorbic acid-2 phosphate and 10 ng/ml TGF-β1. To investigate the effect of different metabolic substrates, other groups were exposed to additional 25 mM fructose. The media were replaced every second day until day 21 when all the pellets were harvested for further analyses. Biochemical analysis for glycosaminoglycans into pellets and released in medium was performed using the DMMB method. Expression of GLUT3 and GLUT5 was assayed by qPCR and validated using FACS analysis and immunofluorescence in monolayer cultures. Chondrogenic differentiation was further confirmed by qPCR analysis of COL2A1, COL1A1, COL10A1, ACAN, RUNX2, SOX9, SP7, MMP13, and PPARG, normalized on RPLP0. Type 2 collagen expression was subsequently validated by immunofluorescence analysis.ResultsWe show for the first time the presence of fructose transporter GLUT5 in BMSC and its regulation during chondrogenic commitment. Additionally, decreasing glucose concentration during chondrogenesis dramatically decreased the yield of differentiation. However, the use of fructose alone or together with low glucose concentrations does not limit cell differentiation, but on the contrary it might help in maintaining a stable chondrogenic phenotype comparable with the standard culture conditions (high glucose).ConclusionThis study provides evidence that BMSC express GLUT5 and differentially regulate GLUT3 in the presence of glucose variation. This study gives a better comprehension of BMSCs sugar use during chondrogenesis.

GAS6 Promotes Robust Expansion of Human Hematopoietic Stem and Progenitor Cells with High Serial Engraftment Activity

Hematopoietic stem cell transplantation is a curative therapy for over 30 hematopoietic illnesses. The inability to sufficiently expand functional HSCs in cultures has partly hampered their therapeutic potential and limited molecular understanding of HSCs. Stem cell agonists (SCA) such as UM171 and StemReginin1 (SR1) have been shown to promote HSC expansion. Recently, we discovered that Ascorbic acid-2-phosphate (AA2P) can act as a novel SCA. Furthermore, using mathematical modeling we formulated a series of stem cell agonist cocktails (SCACs) composed of varying concentrations of 4 SCAs. SCACs promote robust expansion of HSPCs and limited dilution transplant assay revealed that the lead cocktail X2A promoted 15- and 4-fold expansions of scid repopulating cells, when compared to non-cultured cells or cells expanded with the combination of UM171 and SR1 (Manesia et al., 2019). The objective of the present study was to characterize the molecular programs supporting the strong expansion of HSCs in X2A cultures. RNA-Seq data from SCACs expanded CD34+CD45RA- cells revealed the upregulation of several receptor tyrosine kinases (RTKs) including AXL, but also its ligand GAS6 preferentially with X2A. AXL was recently shown to be essential for the self-renewal of leukemia initiating cells. Single cell RNA-seq analyses of CD34+ cells was done to characterize the graft composition of SCACs expanded HSPCs. These analyses revealed that AXL expression is enriched within CD34+CD45RA-CD90+CD49f+ HSC-enriched clusters. Image-flowcytometry analyses confirmed that HSC-enriched cell populations respond to GAS6-activation, resulting in the activation of ERK1/2 downstream of AXL activation. Epigenetic analysis revealed that GAS6/AXL upregulation is regulated by AA2P through DNA-demethylation in a TET dependent manner. Using genetic knockdown, chemical inhibition and GAS6 neutralizing antibody approaches we confirmed that GAS6 activation of AXL drives human HSPC expansion within X2A. In addition, supplementation of SCAC or single SCA-based cultures with GAS6 significantly enhanced the expansion of HSPC subsets with little induction of differentiation (e.g., 640- Vs. 1975-fold for CD34+CD45RA- HSPC in X2A and X2A+GAS6 cultures, respectively (n=3, p<0.05)). Transplant assays with day-14 expanded HSPC (progeny of 850 CD34+ starting cells/mouse) revealed that GAS6 supplementation in X2A cultures increased ensuing engraftment by 10-fold in both primary and secondary NSG mice (n=2, p<0.0001). For instance, the % of human CD45+ BM cells in secondary mice were of 2.3±0.1% and 25.7±1.3% in X2A and X2A+GAS6 groups. Remarkably, the net numbers of human colony-forming unit and CD34+ BM cells were increased by 7- and 15-fold (p<0.0001) in primary, and by 20- and 26-fold (p<0.0001) in secondary transplants by GAS6, respectively. In contrast, GAS6 neutralization in cultures impaired ensuing serial engraftment by 8- and 30-fold respectively Vs. X2A control (p<0.01). Lastly, SCAC and GAS6 were also shown to improve the expansion of adult G-CSF mobilized peripheral blood stem cells. In conclusion, we demonstrate that AA2P-mediated HSPC expansion is driven through DNA demethylation leading to enhanced expression of AXL and its ligand GAS6. We also show for the first time that GAS6 is a potent growth factor that promote robust expansion of human HSPCs including serial engrafting HSCs resulting in unexpectedly high engraftment.

Cerium ions triggered dual-readout immunoassay based on aggregation induced emission effect and 3,3′,5,5′-tetramethylbenzidine for fluorescent and colorimetric detection of ochratoxin A

Ochratoxin A (OTA) is one of the most prevalent and toxic mycotoxins. Ultrasensitive and convenient detection of OTA is urgent demanded for public health. In this work, a dual-readout immunoassay was established for the detection of OTA based on Ce4+ oxidizing 3,3′,5,5′-tetramethylbenzidine (TMB) and Ce3+ inducing aggregation induced emission (AIE) of Au nanoclusters (AuNCs). Under alkaline phosphatase (ALP), the ascorbic acid 2-phosphate (AA2P) can form ascorbic acid (AA) by dephosphorylation. The AA can reduce Ce4+ to generate Ce3+, which induced the AIE of AuNCs to enhance the fluorescence intensity of AuNCs. Meanwhile, unreacted Ce4+ oxidized TMB to form blue oxTMB. Thus, a dual-readout immunoassay was developed based on AIE of AuNCs and TMB as substrate. The limits of detection (LODs) were as low as 0.62 ng/mL for fluorescent assay and 0.81 ng/mL for colorimetric assay. The recoveries of OTA from corn were 94.4%–107.7% for the fluorescent mode and 93.7%–106.9% for the colorimetric mode. The results verified that the cerium ions triggered dual-readout immunoassay was reliable to sensitive detect OTA in corn samples.

A ratiometric fluorescence method based on nitrogen-doped carbon quantum dots for the determination of the activity of alkaline phosphatase.

Herein, a simple and sensitive ratiometric fluorescence sensing platform to detect alkaline phosphatase (ALP) activity is developed on the basis of yellow fluorescent nitrogen-doped carbon quantum dots (YNCDs). The hydrolysis of ascorbic acid 2-phosphate (AAP) into ascorbic acid (AA) is catalyzed by ALP. Then, AA will react with o-phenylenediamine (OPD) to form 3-(1,2-dihydroxyethyl)furo[3,4b]-quinoxaline (QXD) which is a blue fluorescent quinoxaline derivative with emission at 435nm in the presence of Cu2+. YNCDs have yellow fluorescence emission at 555nm, and can maintain stable in QXD reaction system. Therefore, by utilizing the fluorescence of YNCDs at 555nm as reference signal and the fluorescence of QXD at 435nm as report signal, we can detect the ALP activity by monitoring the fluorescence ratio (F435/F555). The linear range is 0.5-5 U/L, and the limit of detection is 0.14 U/L. An application of this method for the analysis of ALP in human serum has given satisfactory results. A ratiometric fluorescent nanoprobe for ascorbic acid and alkaline phosphatase detection with excellent biocompatible and high sensitivity was successfully constructed based on YNCDs and QXD.

A smartphone-integrated colorimetric quantitative analysis platform based on oxidase-like Ce(IV)-ATP-Tris CPNs/CNF test strip for detection of inorganic arsenic in rice

Herein, a colorimetric sensing system based on cerium(IV) coordination polymer nanoparticles (Ce(IV)-ATP-Tris CPNs) was proposed for As(V) detection. Ce(IV)-ATP-Tris CPNs show excellent oxidase-like activity, triggering 3,3′,5,5′-tetramethylbenzidine (TMB) chromogenic reaction. With addition of ascorbic acid 2-phosphate (AAP) and acid phosphatase (ACP), ACP can hydrolyze AAP to produce antioxidative ascorbic acid (AA), inhibiting TMB chromogenic reaction. After that, introduction of As(V) can inhibit ACP, recovering TMB chromogenic reaction. Therefore, sensitive and selective As(V) detection is achieved. Moreover, Ce(IV)-ATP-Tris CPNs were transformed into cellulose nanofiber (CNF) to form test strip (Ce(IV)-ATP-Tris CPNs/CNF). Inorganic arsenic in rice can be detected by test strip, color of that can be measured by smartphone-integrated colorimetric quantitative analysis platform. Given this, rapid and convenient strip test of inorganic arsenic in rice by using this platform was achieved. Hence, this platform possesses great application potential in the field of inorganic arsenic in rice, even food safety.

Photoelectrochemical biosensor based on CdS quantum dots anchored h-BN nanosheets and tripodal DNA walker for sensitive detection of miRNA-141

Herein, a high-sensitivity photoelectrochemical (PEC) biosensor was developed based on CdS quantum dots (QDs) sensitized porous hexagonal boron nitride (h-BN) nanosheets (NSs) and the multiple sites tripodal DNA walker (TDW) formed by catalytic hairpin assembly (CHA). Noticeably, the porous structure of h-BN NSs gives it a lasting gift of large specific surface areas and extensive active reaction sites, which makes it possible to be employed as photoelectric substrate material. The h-BN/CdS QDs composite material promotes the transmission of photogenerated electrons and holes, the outstanding photoelectric conversion efficiency. Meanwhile, the CHA-formed TDWs triggered by miRNA-141 moved on track strand-functionalized electrode, so that a large number of alkaline phosphatase (ALP) was immobilized on the electrode surface and further in situ catalyzed ascorbic acid 2-phosphate (AAP) to produce ascorbic acid (AA) as the electron donor. As the result of the decisive influence of electron donor on PEC biosensor, the sensitive detection of miRNA-141 was realized. The proposed PEC biosensor displayed an excellent linear relationship ranging from 1 fM to 100 nM with a detection limit of 0.73 fM, providing a powerful strategy for early clinical diagnosis and biomedical research.

Effect of ascorbic acid and tri-sodium phosphate treatment at different packaging conditions on cold storage Bagrus bayad fish quality

This research paper aimed at evaluating the influence of ascorbic acid (AA) and tri-sodium phos¬phate (TSP) under different packaging condition on improving the shelf life and qualities of fillets of Bagrus bayad cold fish at 4±1°C for 12 days. The microbiological analysis performed were the total bacterial count (TBC), proteolytic bacterial count (PBC), lipolytic bacterial count (LBC), Coliform count (CC), Staphylococcus aureus (SA), Salmonella &amp; Shigella (SS) and yeast &amp; mold (YM). The obtained results indicated that all noticed analysis parameters (TBC, PBC, LBC, CC, SA, SS, YM, AA and TSP).were gradually increased during storage period in different treatments. The increases of these parameters were significantly higher (p &lt;0.05) in control sample than samples treatment with (AA) and (TSP). The obtained results also showed that there was a significant (p&lt;0.05) extension of the shelf-life through delaying the microbial growth of the samples treated with (TSP) and (AA), respec¬tively. In addition, the shelf-life and the storage period for these products were found to be less than 3 days for control sample and more than 12 days of samples treated with (TSP) under cold storage (4°C). Therefore, the (TSP) had a high effectiveness for extending the shelf-life and enhancing quality attri¬butes of raw fish fillets during cold storage.

Fluorescence immunoassay based on alkaline phosphatase-induced in situ generation of fluorescent non-conjugated polymer dots

Alkaline phosphatase (ALP) activity assay is not only significant to the clinical diagnosis of some related disease, but also momentous to the construction of ALP-based enzyme-linked immunosorbent assay (ELISA). Herein, for the first time, we have discovered that ascorbic acid (AA) can specially react with N-methylethylenediamine (N-MEDA) to generate fluorescent non-conjugated polymer dots (NCPDs) under mild conditions. On the basis of the AA-responsive emission and ALP-catalyzed hydrolysis of ascorbic acid 2-phosphate (AA2P) to AA, we have exploited a fluorometric ALP activity assay with high sensitivity and selectivity. Furthermore, by means of conventional ALP-based ELISA platform, a conceptual fluorescent ELISA has been constructed and applied in the potential clinical diagnosis, during which cardiac troponin I (cTnI), a well-established biomarker of acute myocardial infarction, has been chosen as the model target. We envision that such original fluorescent NCPDs generation-enabled ELISA could become a versatile tool in biochemical sensing and medical diagnosis in the future.

Fluorescence dual “turn-on” detection of acid phosphatase via fluorescence resonance energy transfer and dual quenching strategy to improve sensitivity

A fluorescent dual “turn-on” method is utilized to detect acid phosphatase (ACP) via dual quenching and fluorescence resonance energy transfer (FRET) strategy to improve sensitivity for the first time. Bovine serum albumin protected gold nanoclusters (BSA-AuNCs) are utilized as fluorescence probe and peroxidase-mimicking catalyst. BSA-AuNCs oxidize 3,3′,5,5′-tetramethylbenzidine (TMB) to TMB (oxTMB) with the absorbance peak at 652 nm. Fluorescence spectrum of BSA-AuNCs with emission peak at 635 nm is largely overlapped with absorption spectrum of oxTMB, which brings FRET from BSA-AuNCs to oxTMB and fluorescence is quenched. When Ce3+ is added, BSA-AuNCs catalyze the oxidation of Ce3+ to Ce4+ by H2O2. Ce4+ has oxidizing properties, which can oxidize TMB into oxTMB and enhance FRET. It achieves the fluorescence is quenched further more. This dual fluorescence quenching effect brings a low background for ACP detection. ACP catalyzes ascorbic acid 2-phosphate (AA2P) hydrolysis into phosphate ion (PO43−) and ascorbic acid (AA). Owing to high coordination between Ce3+ and PO43−, TMB oxidation is prevented and fluorescent is turned-on. AA reduces oxTMB into TMB, which makes FRET be prevented and fluorescence is also increased. Therefore it is utilized in ACP detection from 0.01 to 2 U/L with high selectivity and accuracy.

Sensitive and selective electrochemical determination of doxycycline in pharmaceutical formulations using poly(dipicrylamine) modified glassy carbon electrode

A voltammetric sensor was developed by electropolymerization of 2, 4, 6–2′, 4′, 6′ hexanitrodiphenylamine (dipicrylamine) on a glassy carbon electrode. Poly(dipicrylamine) modified glassy carbon electrode (poly(DPA)/GCE) was characterized with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The electrochemical behavior of doxycycline (DOX) in phosphate buffer solution (PBS) of pH 8.0 was investigated at the surface of poly(DPA)/GCE. Compared with glassy carbon electrode, doxycycline showed two well-resolved irreversible oxidative peaks (Ipa1 and Ipa2) at the poly(DPA)/GCE. Under optimized square wave voltammetric conditions, peaks current at poly(DPA)/GCE showed good linearity with the concentration of DOX in the range of 5–200 μM with a detection limit (LOD) of 0.15 μM and 0.13 μM for Ipa1 and Ipa2, respectively. Besides its high stability and reproducibility, the response of DOX at poly(DPA)/GCE was not affected in the presence of glucose, ascorbic acid and chloroquine phosphate. The developed sensor has been successfully applied for the detection of DOX in four brands of pharmaceutical samples and the detected amount of DOX in each brand was above 90.13% of the labeled value.

Enhanced angiogenic potential of adipose-derived stem cell sheets by integration with cell spheroids of the same source

BackgroundAdipose-derived stem cell (ASC) has been considered as a desirable source for cell therapy. In contrast to combining scaffold materials with cells, ASCs can be fabricated into scaffold-free three-dimensional (3D) constructs to promote regeneration at tissue level. However, previous reports have found decreased expression of vascular endothelial growth factor (VEGF) in ASC sheets. In this study, we aimed to integrate ASC spheroids into ASC sheets to enhance the angiogenic capability of cell sheets.MethodsASCs were seeded in agarose microwells to generate uniform cell spheroids with adjustable size, while extracellular matrix deposition could be stimulated by ascorbic acid 2-phosphate to form ASC sheets. RNA sequencing was performed to identify the transcriptomic profiles of ASC spheroids and sheets relative to monolayer ASCs. By transferring ASC spheroids onto ASC sheets, the spheroid sheet composites could be successfully fabricated after a short-term co-culture, and their angiogenic potential was evaluated in vitro and in ovo.ResultsRNA sequencing analysis revealed that upregulation of angiogenesis-related genes was found only in ASC spheroids. The stimulating effect of spheroid formation on ASCs toward endothelial lineage was demonstrated by enhanced CD31 expression, which maintained after ASC spheroids were seeded on cell sheets. Relative to ASC sheets, enhanced expression of VEGF and hepatocyte growth factor was also noted in ASC spheroid sheets, and conditioned medium of ASC spheroid sheets significantly enhanced tube formation of endothelial cells in vitro. Moreover, chick embryo chorioallantoic membrane assay showed a significantly higher capillary density with more branch points after applying ASC spheroid sheets, and immunohistochemistry also revealed a significantly higher ratio of CD31-positive area.ConclusionIn the spheroid sheet construct, ASC spheroids can augment the pro-angiogenesis capability of ASC sheets without the use of exogenous biomaterial or genetic manipulation. The strategy of this composite system holds promise as an advance in 3D culture technique of ASCs for future application in angiogenesis and regeneration therapies.

Fluorescence recovery based on synergetic effect for ALP detection

Alkaline phosphatase (ALP) is an important biomarker associated with diabetes, liver dysfunction, bone diseases, and breast cancer. Here we developed a method based on synergetic fluorescence recovery for the sensitive detection of ALP. Cadmium-zinc-selenium (CdZnSe) quantum dots (QDs) were prepared by one-pot water bath method without any complicated and rigorous conditions. CdZnSe QDs displayed high luminous efficiency, good stability, and good biocompatibility. KMnO4 and ascorbic acid phosphate (AAP) can dynamically quench the fluorescence of CdZnSe QDs. Ascorbic acid, produced by ALP-catalyzed hydrolysis of AAP, reacted with KMnO4, causing the synergetic fluorescence recovery of CdZnSe QDs. The synergetic recovery efficiency correlates well with the logarithmic ALP concentration in the range of 2.5–250 U/L with a detection limit of 0.21 U/L. In addition, good recoveries were obtained in the detection of ALP in human serum. This method provided a new research idea to improve the detection sensitivity and selectivity of ALP detection.

MOFs-functionalized regenerable SERS sensor based on electrochemistry for pretreatment-free detection of serum alkaline phosphatase activity

Alkaline phosphatase (ALP), widely distributing in the human body, associates with many liver and bone diseases. Herein, we designed a reusable surface-enhanced Raman scattering (SERS) sensor via electrochemical self-assembly of Au nanoparticles (AuNPs) and zinc-based metal organic frameworks (Zn-MOFs) at anodic aluminum oxide (AAO) membrane for detecting ALP activity. The detection is based on the sensitive SERS response of the newly synthesized 2-mercaptoquinone (2-MBQ) modified on AuNPs surface towards ascorbic acid (AA) formed from the dephosphorylation of ascorbic acid 2-phosphate (AA2P) in the presence of ALP. Benefitting from the sieving function of Zn-MOFs and superiority of SERS, the developed sensor exhibited strong anti-interference performance and wide detection range from 1 to 300 U/L with a detection limit of 0.38 U/L, which allowed the direct detection of ALP activity in serum without any pretreatment. Besides, we regenerated the used sensor through the electrochemical oxidation of the reduced probe molecule 2-mercaptohydroquinone (2-MHQ). The regenerated sensor exhibited good maintenance of sensitive response to ALP, in favor of improving the usage efficiency and cutting the test cost. This work provides a promising platform for early diagnosis of ALP-related diseases and paves the way towards developing regenerable and pretreatment-free SERS sensors.

Synergistic effect-mediated fluorescence switching of nitrogen-doped carbon dots for visual detection of alkaline phosphatase

The natural enzyme alkaline phosphatase (ALP), which is associated with a large number of diseases, plays an essential role in clinicopathological examinations. Therefore, it is of great significance to quantify the ALP activity levels. In this work, a switchable fluorescence assay of detecting alkaline phosphatase (ALP) activity was proposed using Fe3+ to adjust the fluorescence of nitrogen-doped carbon dots (CDs). Wherein, CDs chelated with Fe3+, leading to a remarkable and dynamic fluorescence quenching. However, ascorbic acid (AA) and phosphate group (Pi), resulting from the enzymatic reaction between ascorbic acid-2-phosphate (AAP) and ALP, enabled the great fluorescence recovery, which was possibly related with the synergistic effect including the chemical redox of AA towards Fe3+ and the formation of a stable FePO4 complex. Based on this synergistic effect-mediated switching mechanism, the indirect detection of ALP activity was developed in the range of 15–445 U/L with the limit of detection at 2.576 U/L (3σ/k) and the limit of quantitation at 8.587 U/L (10σ/k), which could be observed by the naked-eyes. Then, the proposed method was further applied to the detection of ALP activity in human serum and urine samples with satisfactory results, which was in good agreement with the result obtained by ALP commercial kit. This proposed synergistic effect from AA and Pi provides a new insight for ALP activity detection.

Enzyme-controllable just-in-time production system of copper hexacyanoferrate nanoparticles with oxidase-mimicking activity for highly sensitive colorimetric immunoassay

Nanozymes are a series of elaborately designed nanomaterials that can mimic the catalytic sites of natural enzymes for reactions. Bypassing the tedious design and preparation of nanomaterial, in this work, we report on a novel just-in-time production system of copper hexacyanoferrate nanoparticles (CHNPs), which act as an oxidase-mimicking nanozyme. This system can rapidly produce CHNPs nanozyme on demand by simply mixing Cu(II) with potassium hexacyanoferrate(III) (K3[Fe(CN)6]). It is found that once K3[Fe(CN)6] is reduced to K4[Fe(CN)6], the formation of CHNPs is inhibited. Therefore, the just-in-time production system of CHNPs was coupled with alkaline phosphatase (ALP) to construct an enzyme-controllable just-in-time production (ECJP) system, in which ALP could inhibit the production of by catalyzing the hydrolysis of ascorbic acid 2-phosphate (AAP) to generating ascorbic acid (AA). The ECJP system is then used to probe the activity of ALP by employing 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) as the chromogenic substrate, and a detection limit of 0.003 U L−1 was achieved. Moreover, by adapting ALP as the enzyme label, an ECJP system-based colorimetric immunoassay protocol was established for sensitive detection of aflatoxin B1 (AFB1), and a detection limit as low as 0.73 pg mL−1 was achieved. The developed immunoassay method is successfully applied to the detection of AFB1 in peanut samples. The operation of ECJP system is quite simple and the coupling of ALP with CHNPs nanozyme can arouse dual enzyme-like cascade signal amplification. So, we believe this work can offer a new perspective for the development of nanozymes-based biodetection methods and colorimetric immunoassay strategies.

Efficient Biocatalytic System for Biosensing by Combining Metal-Organic Framework (MOF)-Based Nanozymes and G-Quadruplex (G4)-DNAzymes.

A high catalytic efficiency associated with a robust chemical structure are among the ultimate goals when developing new biocatalytic systems for biosensing applications. To get ever closer to these goals, we report here on a combination of metal-organic framework (MOF)-based nanozymes and a G-quadruplex (G4)-based catalytic system known as G4-DNAzyme. This approach aims at combining the advantages of both partners (chiefly, the robustness of the former and the modularity of the latter). To this end, we used MIL-53(Fe) MOF and linked it covalently to a G4-forming sequence (F3TC), itself covalently linked to its cofactor hemin. The resulting complex (referred to as MIL-53(Fe)/G4-hemin) exhibited exquisite peroxidase-mimicking oxidation activity and an excellent robustness (being stored in water for weeks). These properties were exploited to devise a new biosensing system based on a cascade of reactions catalyzed by the nanozyme (ABTS oxidation) and an enzyme, the alkaline phosphatase (or ALP, ascorbic acid 2-phosphate dephosphorylation). The product of the latter poisoning the former, we thus designed a biosensor for ALP (a marker of bone diseases and cancers), with a very low limit of detection (LOD, 0.02 U L-1), which is operative in human plasma samples.

One-Step Determination of Alkaline Phosphatase in Human Serum Based on Manganese (IV) Dioxide/Manganese (II)-Mediated Nuclear Magnetic Resonance (NMR) Relaxation

Alkaline phosphatase (ALP) is a significant biomarker of diseases, such as osteoblastic bone cancer, hepatitis, and myeloma, and hence its determination is essential and important. MnO2/Mn2+ conversion-based magnetic relaxation sensing was developed to directly determine ALP in blood. In the presence of ALP, 2-phospho-L-ascorbic acid trisodium salt was catalyzed to produce ascorbic acid (AA), which reduced MnO2 to Mn2+, causing transverse relaxation time (T2) decay. A linear relationship of ALP from 5 mU·L−1 to 500 mU·L−1 with T2 signal and a low limit of detection (LOD) of 3.32 mU·L−1 were obtained using the optimized conditions of 200 µM of ascorbic acid-phosphate (AAP), 10 µg·mL−1 of MnO2, 37 °C, pH 7.4, and a 30 min reaction time. When directly measuring ALP in serum with the proposed method, the recoveries were from 99.2% to 101.3%, in accordance with values obtained by a commercial assay. In conclusion, a sensitive one-step method for ALP in complex samples was established which has potential for clinical applications.