Journal of Pharmaceutical Analysis

2022, 12(4): ii-ii. doi: 10.1016/S2095-1779(22)00064-8

Abstract(169) HTML Full Text PDF(3)

Abstract:

Corrigendum to “The effective transfection of a low dose of negatively charged drug-loaded DNA-nanocarriers into cancer cells via scavenger receptors”[Journal of Pharmaceutical Analysis volume 11 (2021) 174-182]

Mirza Muhammad Faran Ashraf Baig, Chengfei Zhang, Muhammad Furqan Akhtar, Ammara Saleem, Jahanzeb Mudassir

2022, 12(4): 698-700. doi: 10.1016/j.jpha.2022.05.002

Abstract(146) HTML Full Text PDF(4)

Abstract:

Current developments of bioanalytical sample preparation techniques in pharmaceuticals

Rahul G. Ingle, Su Zeng, Huidi Jiang, Wei-Jie Fang

2022, 12(4): 517-529. doi: 10.1016/j.jpha.2022.03.001

Abstract(409) HTML Full Text PDF(41)

Abstract:
Sample preparation is considered as the bottleneck step in bioanalysis because each biological matrix has its own unique challenges and complexity. Competent sample preparation to extract the desired analytes and remove redundant components is a crucial step in each bioanalytical approach. The matrix effect is a key hurdle in bioanalytical sample preparation, which has gained extensive consideration. Novel sample preparation techniques have advantages over classical techniques in terms of accuracy, automation, ease of sample preparation, storage, and shipment and have become increasingly popular over the past decade. Our objective is to provide a broad outline of current developments in various bioanalytical sample preparation techniques in chromatographic and spectroscopic examinations. In addition, how these techniques have gained considerable attention over the past decade in bioanalytical research is mentioned with preferred examples. Modern trends in bioanalytical sample preparation techniques, including sorbent-based microextraction techniques, are primarily emphasized.

Recent advances in ultrasound-controlled fluorescence technology for deep tissue optical imaging

Rui-Lin Liu, Ru-Qian Cai

2022, 12(4): 530-540. doi: 10.1016/j.jpha.2021.10.002

Abstract(218) HTML Full Text PDF(7)

Abstract:
Fluorescence imaging is a noninvasive and dynamic real-time imaging technique; however, it exhibits poor spatial resolution in centimeter-deep tissues because biological tissues are highly scattering media for optical radiation. The recently developed ultrasound-controlled fluorescence (UCF) imaging is a novel imaging technique that can overcome this bottleneck. Previous studies suggest that the effective contrast agent and sensitive imaging system are the two pivotal factors for generating high-resolution UCF images ex vivo and/or in vivo. Here, this review highlights the recent advances (2015-2020) in the design and synthesis of contrast agents and the improvement of imaging systems to realize high-resolution UCF imaging of deep tissues. The imaging performances of various UCF systems, including the signal-to-noise ratio, imaging resolution, and imaging depth, are specifically discussed. In addition, the challenges and prospects are highlighted. With continuously increasing research interest in this field and emerging multidisciplinary applications, UCF imaging with higher spatial resolution and larger imaging depth may be developed shortly, which is expected to have a far-reaching impact on disease surveillance and/or therapy.

Interactions between gut microbiota and berberine, a necessary procedure to understand the mechanisms of berberine

Hao Cheng, Juan Liu, Yuzhu Tan, Wuwen Feng, Cheng Peng

2022, 12(4): 541-555. doi: 10.1016/j.jpha.2021.10.003

Abstract(571) HTML Full Text PDF(31)

Abstract:
Berberine (BBR), an isoquinoline alkaloid, has been found in many plants, such as Coptis chinensis Franch and Phellodendron chinense Schneid. Although BBR has a wide spectrum of pharmacological effects, its oral bioavailability is extremely low. In recent years, gut microbiota has emerged as a cynosure to understand the mechanisms of action of herbal compounds. Numerous studies have demonstrated that due to its low bioavailability, BBR can interact with the gut microbiota, thereby exhibiting altered pharmacological effects. However, no systematic and comprehensive review has summarized these interactions and their corresponding influences on pharmacological effects. Here, we describe the direct interactive relationships between BBR and gut microbiota, including regulation of gut microbiota composition and metabolism by BBR and metabolization of BBR by gut microbiota. In addition, the complex interactions between gut microbiota and BBR as well as the side effects and personalized use of BBR are discussed. Furthermore, we provide our viewpoint on future research directions regarding BBR and gut microbiota. This review not only helps to explain the mechanisms underlying BBR activity but also provides support for the rational use of BBR in clinical practice.

18beta-glycyrrhetinic acid induces ROS-mediated apoptosis to ameliorate hepatic fibrosis by targeting PRDX1/2 in activated HSCs

Qian Zhang, Piao Luo, Liuhai Zheng, Jiayun Chen, Junzhe Zhang, Huan Tang, Dandan Liu, Xueling He, Qiaoli Shi, Liwei Gu, Jiahao Li, Qiuyan Guo, Chuanbin Yang, Yin Kwan Wong, Fei Xia, Jigang Wang

2022, 12(4): 570-582. doi: 10.1016/j.jpha.2022.06.001

Abstract(602) HTML Full Text PDF(46)

Abstract:
Hepatic stellate cells (HSCs) are essential drivers of fibrogenesis. Inducing activated-HSC apoptosis is a promising strategy for treating hepatic fibrosis. 18beta-glycyrrhetinic acid (18β-GA) is a natural compound that exists widely in herbal medicines, such as Glycyrrhiza uralensis Fisch, which is used for treating multiple liver diseases, especially in Asia. In the present study, we demonstrated that 18β-GA decreased hepatic fibrosis by inducing the apoptosis in activated HSCs. 18β-GA inhibited the expression of α-smooth muscle actin and collagen type I alpha-1. Using a chemoproteomic approach derived from activity-based protein profiling, together with cellular thermal shift assay and surface plasmon resonance, we found that 18β-GA covalently targeted peroxiredoxin 1 (PRDX1) and peroxiredoxin 2 (PRDX2) proteins via binding to active cysteine residues and thereby inhibited their enzymatic activities. 18β-GA induced the elevation of reactive oxygen species (ROS), resulting in the apoptosis of activated HSCs. PRDX1 knockdown also led to ROS-mediated apoptosis in activated HSCs. Collectively, our findings revealed the target proteins and molecular mechanisms of 18β-GA in ameliorating hepatic fibrosis, highlighting the future development of 18β-GA as a novel therapeutic drug for hepatic fibrosis.

Covalent organic nanospheres as a fiber coating for solid-phase microextraction of genotoxic impurities followed by analysis using GC-MS

Yanfang Zhao, Jingkun Li, Hanyi Xie, Huijuan Li, Xiangfeng Chen

2022, 12(4): 583-589. doi: 10.1016/j.jpha.2021.12.002

Abstract(182) HTML Full Text PDF(10)

Abstract:
Covalent organic nanospheres (CONs) were explored as a fiber coating for solid-phase microextraction of genotoxic impurities (GTIs) from active ingredients (AIs). CONs were synthesized by an easy solution-phase procedure at 25℃. The obtained nanospheres exhibited a high specific surface area, good thermostability, high acid and alkali resistance, and favorable crystallinity and porosity. Two types of GTIs, alkyl halides (1-iodooctane, 1-chlorobenzene, 1-bromododecane, 1,2-dichlorobenzene, 1-bromooctane, 1-chlorohexane, and 1,8-dibromooctane) and sulfonate esters (methyl p-toluenesulfonate and ethyl p-toluenesulfonate), were chosen as target molecules for assessing the performance of the coating. The prepared coating achieved high enhancement factors (5097-9799) for the selected GTIs. The strong affinity between CONs and GTIs was tentatively attributed to π-π and hydrophobicity interactions, large surface area of the CONs, and size-matching of the materials. Combined with gas chromatography-mass spectrometry (GC-MS), the established analytical method detected the GTIs in capecitabine and imatinib mesylate samples over a wide linear range (0.2-200 ng/g) with a low detection limit (0.04-2.0 ng/g), satisfactory recovery (80.03%-109.5%), and high repeatability (6.20%-14.8%) and reproducibility (6.20%-14.1%). Therefore, the CON-coated fibers are promising alternatives for the sensitive detection of GTIs in AI samples.

Metabolomic fingerprinting of porcine lung tissue during pre-clinical prolonged ex vivo lung perfusion using in vivo SPME coupled with LC-HRMS

Nikita Looby, Anna Roszkowska, Aadil Ali, Barbara Bojko, Marcelo Cypel, Janusz Pawliszyn

2022, 12(4): 590-600. doi: 10.1016/j.jpha.2022.06.002

Abstract(192) HTML Full Text PDF(19)

Abstract:
Normothermic ex vivo lung perfusion (NEVLP) has emerged as a modernized organ preservation technique that allows for detailed assessment of donor lung function prior to transplantation. The main goal of this study was to identify potential biomarkers of lung function and/or injury during a prolonged (19 h) NEVLP procedure using in vivo solid-phase microextraction (SPME) technology followed by liquid chromatography-high resolution mass spectrometry (LC-HRMS). The use of minimally invasive in vivo SPME fibers for repeated sampling of biological tissue permits the monitoring and evaluation of biochemical changes and alterations in the metabolomic profile of the lung. These in vivo SPME fibers were directly introduced into the lung and were also used to extract metabolites (on-site SPME) from fresh perfusate samples collected alongside lung samplings. A subsequent goal of the study was to assess the feasibility of SPME as an in vivo method in metabolomics studies, in comparison to the traditional in-lab metabolomics workflow. Several upregulated biochemical pathways involved in pro- and anti-inflammatory responses, as well as lipid metabolism, were observed during extended lung perfusion, especially between the 11th and 12th hours of the procedure, in both lung and perfusate samples. However, several unstable and/or short-lived metabolites, such as neuroprostanes, have been extracted from lung tissue in vivo using SPME fibers. On-site monitoring of the metabolomic profiles of both lung tissues through in vivo SPME and perfusate samples on site throughout the prolonged NEVLP procedure can be effectively performed using in vivo SPME technology.

Simultaneous determination of LY3214996, abemaciclib, and M2 and M20 metabolites in human plasma, cerebrospinal fluid, and brain tumor by LC-MS/MS

Tigran Margaryan, Mackenna Elliott, Nader Sanai, Artak Tovmasyan

2022, 12(4): 601-609. doi: 10.1016/j.jpha.2022.05.003

Abstract(210) HTML Full Text PDF(10)

Abstract:
A sensitive and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method was established for the quantification of total and unbound concentrations of LY3214996, an extracellular signal-regulated kinase inhibitor; abemaciclib, a cyclin-dependent kinase 4/6 inhibitor; and abemaciclib active metabolites, M2 and M20, in human plasma, brain tumor, and cerebrospinal fluid samples. The method was validated over a concentration range of 0.2-500 nM within a total run time of 3.8 min using isocratic elution on a Kinetex^TM F₅ column. Detection was performed on a Sciex QTRAP 6500+ mass spectrometer employing multiple reaction monitoring mode under positive electrospray ionization. The intra- and inter-batch accuracy as well as the precision of the method for all matrices was within ±20% and ≤ 20% at the lower limit of quantification, and within ±15% and ≤ 15% for other quality control levels for all analytes. The unbound fractions of drugs and metabolites in spiked and patient samples were determined using an optimized equilibrium dialysis. The validated method was successfully applied in a phase 0/2 clinical trial to assess the central nervous system penetration of LY3214996 and abemaciclib.

In situ synthesis of a spherical covalent organic framework as a stationary phase for capillary electrochromatography

Ning He, Zhentao Li, Changjun Hu, Zilin Chen

2022, 12(4): 610-616. doi: 10.1016/j.jpha.2022.06.005

Abstract(312) HTML Full Text PDF(11)

Abstract:
Covalent organic frameworks (COFs) are a novel type of crystalline porous organic polymer materials recently developed. It has several advantages in chromatographic separation field, such as high thermal stability, porosity, structural regularity, and large specific surface area. Here, a novel spherical COF 1,3,5-tris(4-aminophenyl)benzene (TAPB) and 2,5-bis(2-propyn-1-yloxy)-1,4-benzenedicarboxaldehyde (BPTA) was developed as an electrochromatographic stationary phase for capillary electrochromatography separation. The COF TAPB-BPTA modified capillary column was fabricated via a facile in situ growth method at room temperature. The characterization results of scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy, and X-ray diffraction (XRD) confirmed that COF TAPB-BPTA were successfully modified onto the capillary inner surface. The electrochromatography separation performance of the COF TAPB-BPTA modified capillary was investigated. The prepared column demonstrated outstanding separation performance toward alkylbenzenes, phenols, and chlorobenzenes compounds. Furthermore, the baseline separations of non-steroidal anti-inflammatory drugs (NSAIDs) and parabens with good efficiency and high resolution were achieved. Also, the prepared column possessed satisfactory precision of the intra-day runs (n=5), inter-day runs (n=3), and parallel columns (n=3), and the relative standard deviations (RSDs) of the retention times of tested alkylbenzenes were all less than 2.58%. Thus, this new COF-based stationary phase shows tremendous application potential in chromatographic separation field.

Preparation of Fe₃O₄@SW-MIL-101-NH₂ for selective pre-concentration of chlorogenic acid metabolites in rat plasma, urine, and feces samples

Shi-Jun Yin, Xi Zhou, Li-Jing Peng, Fang Li, Guo-Can Zheng, Feng-Qing Yang, Yuan-Jia Hu

2022, 12(4): 617-626. doi: 10.1016/j.jpha.2022.01.002

Abstract(231) HTML Full Text PDF(14)

Abstract:
An innovative sandwich-structural Fe-based metal-organic framework magnetic material (Fe₃O₄@SW-MIL-101-NH₂) was fabricated using a facile solvothermal method. The characteristic properties of the material were investigated by field emission scanning electron microscopy, transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, X-ray powder diffraction, vibrating sample magnetometry, and Brunauer-Emmett-Teller measurements. Fe₃O₄@SW-MIL-101-NH₂ is associated with advantages, such as robust magnetic properties, high specific surface area, and satisfactory storage stability, as well as good selective recognition ability for chlorogenic acid (CA) and its metabolites via chelation, hydrogen bonding, and π-interaction. The results of the static adsorption experiment indicated that Fe₃O₄@SW-MIL-101-NH₂ possessed a high adsorption capacity toward CA and its isomers, cryptochlorogenic acid (CCA) and neochlorogenic acid (NCA), and the adsorption behaviors were fitted using the Langmuir adsorption isotherm model. Then, a strategy using magnetic solid-phase extraction (MSPE) and ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF MS/MS) was developed and successfully employed for the selective pre-concentration and rapid identification of CA metabolites in rat plasma, urine, and feces samples. This work presents a prospective strategy for the synthesis of magnetic adsorbents and the high-efficiency pretreatment of CA metabolites.

A highly sensitive bio-barcode immunoassay for multi-residue detection of organophosphate pesticides based on fluorescence anti-quenching

Lingyuan Xu, Xiuyuan Zhang, A.M. Abd El-Aty, Yuanshang Wang, Zhen Cao, Huiyan Jia, J.-Pablo Salvador, Ahmet Hacimuftuoglu, Xueyan Cui, Yudan Zhang, Kun Wang, Yongxin She, Fen Jin, Lufei Zheng, Baima Pujia, Jing Wang, Maojun Jin, Bruce D. Hammock

2022, 12(4): 637-644. doi: 10.1016/j.jpha.2022.05.004

Abstract(308) HTML Full Text PDF(16)

Abstract:
Balancing the risks and benefits of organophosphate pesticides (OPs) on human and environmental health relies partly on their accurate measurement. A highly sensitive fluorescence anti-quenching multi-residue bio-barcode immunoassay was developed to detect OPs (triazophos, parathion, and chlorpyrifos) in apples, turnips, cabbages, and rice. Gold nanoparticles were functionalized with monoclonal antibodies against the tested OPs. DNA oligonucleotides were complementarily hybridized with an RNA fluorescent label for signal amplification. The detection signals were generated by DNA-RNA hybridization and ribonuclease H dissociation of the fluorophore. The resulting fluorescence signal enables multiplexed quantification of triazophos, parathion, and chlorpyrifos residues over the concentration range of 0.01-25, 0.01-50, and 0.1-50 ng/mL with limits of detection of 0.014, 0.011, and 0.126 ng/mL, respectively. The mean recovery ranged between 80.3% and 110.8% with relative standard deviations of 7.3%-17.6%, which correlate well with results obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The proposed bio-barcode immunoassay is stable, reproducible and reliable, and is able to detect low residual levels of multi-residue OPs in agricultural products.

Enzyme-linked immunosorbent assays for quantification of MMAE-conjugated ADCs and total antibodies in cynomolgus monkey sera

Min Pei, Tingting Liu, Lu Ouyang, Jianhua Sun, Xiaojie Deng, Xiaomin Sun, Wei Wu, Peng Huang, Yi-Li Chen, Xiaorong Tan, Xiaoyue Liu, Peng Zhu, Yongzhen Liu, Deheng Wang, Junliang Wu, Qi Wang, Guifeng Wang, Likun Gong, Qiuping Qin, Chunhe Wang

2022, 12(4): 645-652. doi: 10.1016/j.jpha.2021.11.005

Abstract(306) HTML Full Text PDF(24)

Abstract:
Antibody-drug conjugates (ADCs) are commonly heterogeneous and require extensive assessment of exposure-efficacy and exposure-safety relationships in preclinical and clinical studies. In this study, we report the generation of a monoclonal antibody against monomethyl auristatin E (MMAE) and the development, validation, and application of sensitive and high-throughput enzyme-linked immunosorbent assays (ELISA) to measure the concentrations of MMAE-conjugated ADCs and total antibodies (tAb, antibodies in ADC plus unconjugated antibodies) in cynomolgus monkey sera. These assays were successfully applied to in vitro plasma stability and pharmacokinetic (PK) studies of SMADC001, an MMAE-conjugated ADC against trophoblast cell surface antigen 2 (TROP-2). The plasma stability of SMADC001 was better than that of similar ADCs coupled with PEG4-Val-Cit, Lys (m-dPEG24)-Cit, and Val-Cit linkers. The developed ELISA methods for the calibration standards of ADC and tAb revealed a correlation between serum concentrations and the OD₄₅₀ values, with R² at 1.000, and the dynamic range was 0.3-35.0 ng/mL and 0.2-22.0 ng/mL, respectively; the intra- and inter-assay accuracy bias% ranged from -12.2% to -5.2%, precision ranged from -12.4% to -1.4%, and the relative standard deviation (RSD) was less than 6.6% and 8.7%, respectively. The total error was less than 20.4%. The development and validation steps of these two assays met the acceptance criteria for all addressed validation parameters, which suggested that these can be applied to quantify MMAE-conjugated ADCs, as well as in PK studies. Furthermore, these assays can be easily adopted for development of other similar immunoassays.

Design, synthesis, and evaluation of fluoroquinolone derivatives as microRNA-21 small-molecule inhibitors

Yuan-Yuan Hei, Si Wang, Xiao-Xiao Xi, Hai-Peng Wang, Yuanxu Guo, Minhang Xin, Congshan Jiang, Shemin Lu, San-Qi Zhang

2022, 12(4): 653-663. doi: 10.1016/j.jpha.2021.12.008

Abstract(128) HTML Full Text PDF(6)

Abstract:
MicroRNA-21 (miRNA-21) is highly expressed in various tumors. Small-molecule inhibition of miRNA-21 is considered to be an attractive novel cancer therapeutic strategy. In this study, fluoroquinolone derivatives A1-A43 were synthesized and used as miRNA-21 inhibitors. Compound A36 showed the most potent inhibitory activity and specificity for miRNA-21 in a dual-luciferase reporter assay in HeLa cells. Compound A36 significantly reduced the expression of mature miRNA-21 and increased the protein expression of miRNA-21 target genes, including programmed cell death protein 4 (PDCD4) and phosphatase and tensin homology deleted on chromosome ten (PTEN), at 10 μM in HeLa cells. The Cell Counting Kit-8 assay (CCK-8) was used to evaluate the antiproliferative activity of A36; the results showed that the IC₅₀ value range of A36 against six tumor cell lines was between 1.76 and 13.0 μM. Meanwhile, A36 did not display cytotoxicity in BEAS-2B cells (lung epithelial cells from a healthy human donor). Furthermore, A36 significantly induced apoptosis, arrested cells at the G₀/G₁ phase, and inhibited cell-colony formation in HeLa cells. In addition, mRNA deep sequencing showed that treatment with A36 could generate 171 dysregulated mRNAs in HeLa cells, while the expression of miRNA-21 target gene dual-specificity phosphatase 5 (DUSP5) was significantly upregulated at both the mRNA and protein levels. Collectively, these findings demonstrated that A36 is a novel miRNA-21 inhibitor.

Novel assays for quality evaluation of XueBiJing: Quality variability of a Chinese herbal injection for sepsis management

Xuan Yu, Wei Niu, Ya-Ya Wang, Olajide E. Olaleye, Jia-Nan Wang, Meng-Yuan Duan, Jun-Ling Yang, Rong-Rong He, Zi-Xuan Chu, Kai Dong, Gui-Ping Zhang, Chang-Xiao Liu, Chen Cheng, Chuan Li

2022, 12(4): 664-682. doi: 10.1016/j.jpha.2022.01.001

Abstract(274) HTML Full Text PDF(20)

Abstract:
XueBiJing is an intravenous five-herb injection used to treat sepsis in China. The study aimed to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS)- or liquid chromatography-ultraviolet (LC-UV)-based assay for quality evaluation of XueBiJing. Assay development involved identifying marker constituents to make the assay therapeutically relevant and building a reliable one-point calibrator for monitoring the various analytes in parallel. Nine marker constituents from the five herbs were selected based on XueBiJing's chemical composition, pharmacokinetics, and pharmacodynamics. A selectivity test (for "similarity of response") was developed to identify and minimize interference by non-target constituents. Then, an intercept test was developed to fulfill "linearity through zero" for each analyte (absolute ratio of intercept to C response, <2%). Using the newly developed assays, we analyzed samples from 33 batches of XueBiJing, manufactured over three years, and found small batch-to-batch variability in contents of the marker constituents (4.1%-14.8%), except for senkyunolide I (26.5%).

Discovery of human pancreatic lipase inhibitors from root of Rhodiola crenulata via integrating bioactivity-guided fractionation, chemical profiling and biochemical assay

Li-Juan Ma, Xu-Dong Hou, Xiao-Ya Qin, Rong-Jing He, Hao-Nan Yu, Qing Hu, Xiao-Qing Guan, Shou-Ning Jia, Jie Hou, Tao Lei, Guang-Bo Ge

2022, 12(4): 683-691. doi: 10.1016/j.jpha.2022.04.002

Abstract(524) HTML Full Text PDF(57)

Abstract:
Although herbal medicines (HMs) are widely used in the prevention and treatment of obesity and obesity-associated disorders, the key constituents exhibiting anti-obesity activity and their molecular mechanisms are poorly understood. Recently, we assessed the inhibitory potentials of several HMs against human pancreatic lipase (hPL, a key therapeutic target for human obesity), among which the root-extract of Rhodiola crenulata (ERC) showed the most potent anti-hPL activity. In this study, we adopted an integrated strategy, involving bioactivity-guided fractionation techniques, chemical profiling, and biochemical assays, to identify the key anti-hPL constituents in ERC. Nine ERC fractions (retention time=12.5-35 min), obtained using reverse-phase liquid chromatography, showed strong anti-hPL activity, while the major constituents in these bioactive fractions were subsequently identified using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS/MS). Among the identified ERC constituents, 1,2,3,4,6-penta-O-galloyl-β-d-glucopyranose (PGG) and catechin gallate (CG) showed the most potent anti-hPL activity, with pIC₅₀ values of 7.59 ±0.03 and 7.68 ±0.23, respectively. Further investigations revealed that PGG and CG potently inhibited hPL in a non-competitive manner, with inhibition constant (K_i) values of 0.012 and 0.082 μM, respectively. Collectively, our integrative analyses enabled us to efficiently identify and characterize the key anti-obesity constituents in ERC, as well as to elucidate their anti-hPL mechanisms. These findings provide convincing evidence in support of the anti-obesity and lipid-lowering properties of ERC.

Sensitive detection of alkaline phosphatase based on terminal deoxynucleotidyl transferase and endonuclease IV-assisted exponential signal amplification

Weicong Ye, Longjie Li, Zishan Feng, Bocheng Tu, Zhe Hu, Xianjin Xiao, Tongbo Wu

2022, 12(4): 692-697. doi: 10.1016/j.jpha.2021.09.012

Abstract(179) HTML Full Text PDF(4)

Abstract:
Alkaline phosphatase (ALP) is widely expressed in human tissues. ALP plays an important role in the dephosphorylation of proteins and nucleic acids. Therefore, quantitative analysis of ALP plays a vital role in disease diagnosis and the development of biological detection methods. Terminal deoxynucleotidyl transferase (TdT) catalyzes continuous polymerization of deoxynucleotide triphosphates at the 3'-OH end of single-stranded DNA in the absence of a template. In this study, we developed a highly sensitive and selective method based on TdT and endonuclease IV (Endo IV) to quantify ALP activity. After ALP hydrolyzes the 3'-PO₄ end of the substrate and generates 3'-OH, TdT can effectively elongate the 3'-OH end with deoxynucleotide adenine triphosphate (dATP) and produce a poly A tail, which can be detected by the poly T probes. Endo IV digests the AP site in poly T probes to generate a fluorescent signal and a new 3'-OH end, leading to the generation of exponential fluorescence signal amplification. The substrate for TdT elongation was optimized, and a limit of detection of 4.3×10^-3 U/L was achieved for ALP by the optimized substrate structure. This method can also detect ALP in the cell lysate of a single cell. This work has potential applications in disease diagnosis and biomedical detection.

2022 Vol. 12, No. 4