Machine learning-assisted MALDI-TOF MS toward rapid classification of milk products.

This study established a method for rapid classification of milk products by combining matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis with machine learning techniques. The analysis of 2 different types of milk products was used as an example. To select key variables as potential markers, integrated machine learning strategies based on 6 feature selection techniques combined with support vector machine (SVM) classifier were implemented to screen the informative features and classify the milk samples. The models were evaluated and compared by accuracy, Akaike information criterion (AIC), and Bayesian information criterion (BIC). The results showed the least absolute shrinkage and selection operator (LASSO) combined with SVM performs best, with prediction accuracy of 100 ± 0%, AIC of -360 ± 22, and BIC of -345 ± 22. Six features were selected by LASSO and identified based on the available protein molecular mass data. These results indicate that MALDI-TOF MS coupled with machine learning technique could be used to search for potential key targets for authentication and quality control of food products.

Identification of Radix Bupleuri From Different Geographic Origins Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry and Support Vector Machine Algorithm.

BACKGROUND: The geographic origin of Radix bupleuri is an important factor affecting its efficacy, which needs to be effectively identified. OBJECTIVE: The goal is to enrich and develop the intelligent recognition technology applicable to the identification of the origin of traditional Chinese medicine. METHOD: This article establishes an identification method of Radix bupleuri geographic origin based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and support vector machine (SVM) algorithm. The Euclidean distance method is used to measure the similarity between Radix bupleuri samples, and the quality control chart method is applied to quantitatively describe their quality fluctuation. RESULTS: It is found that the samples from the same origin are relatively similar and mainly fluctuate within the control limit, but the fluctuation range is large, and it is impossible to distinguish the samples from different origins. The SVM algorithm can effectively eliminate the impact of intensity fluctuations and huge data dimensions by combining the normalization of MALDI-TOF MS data and the dimensionality reduction of principal components, and finally achieve efficient identification of the origin of Radix bupleuri, with an average recognition rate of 98.5%. CONCLUSIONS: This newly established approach for identification of the geographic origin of Radix bupleuri has been realized, and it has the advantages of objectivity and intelligence, which can be used as a reference for other medical and food-related research. HIGHLIGHTS: A new intelligent recognition method of medicinal material origin based on MALDI-TOF MS and SVM has been established.

Matrix Factorization-Based Dimensionality Reduction Algorithms─A Comparative Study on Spectroscopic Profiling Data.

Spectroscopic profiling data used in analytical chemistry can be very high-dimensional. Dimensionality reduction (DR) is an effective way to handle the potential "curse of dimensionality" problem. Among the existing DR algorithms, many can be categorized as a matrix factorization (MF) problem, which decomposes the original data matrix X into the product of a low-dimensional matrix W and a dictionary matrix H. First, this paper provides a theoretical reformulation of relevant DR algorithms under a unified MF perspective, including PCA (principal component analysis), NMF (non-negative matrix factorization), LAE (linear autoencoder), RP (random projection), SRP (sparse random projection), VQ (vector quantization), AA (archetypical analysis), and ICA (independent component analysis). From this perspective, an open-sourced toolkit has been developed to integrate all of the above algorithms with a unified API. Second, we made a comparative study on MF-based DR algorithms. In a case study of TOF (time-of-flight) mass spectra, the eight algorithms extracted three components from the original 27,619 features. The results are compared by a set of DR quality metrics, e.g., reconstruction error, pairwise distance/ranking property, computational cost, local and global structure preservations, etc. Finally, based on the case study result, we summarized guidelines for DR algorithm selection. (1) For reconstruction quality, choose ICA. In the case study, ICA, PCA, and NMF have high reconstruction qualities (reconstruction error < 2%), ICA being the best. (2) To keep the pairwise topological structure, choose PCA. PCA best preserves the pairwise distance/ranking property. (3) For edge computing and IoT scenarios, choose RP or SRP if reconstruction is not required and the JL-lemma condition is met. The RP family has the best computational performance in the experiment, almost 10-100 times faster than its peers.

Aptamer-conjugated MoS2 for enrichment and direct detection of small molecules in laser desorption/ionization mass spectrometry.

In this work, MoS2 nanosheets were synthesized by the chemical exfoliation method and then modified with a thiol-terminated aptamer via a simple thiol functionalization route. The as-made nanomaterial was characterized by UV-vis absorption spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, and transmission electron microscopy. By integrating the advantages of MoS2 nanosheets and the recognition ability of aptamers, the functionalized nanomaterial has been successfully employed for simultaneous enrichment and analysis of sulfadimethoxine by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). The aptamer-conjugated MoS2 showed an excellent capture ability to eliminate background signals from the species co-existing in a milk sample. The simplicity of the synthesis method and the excellent performance of aptamer-conjugated MoS2 make it an ideal candidate for application in selective MS analysis of the target analyte from complex samples.

MoS2-Assisted LDI Mass Spectrometry for the Detection of Small Molecules and Quantitative Analysis of Sulfonamides in Serum.

A two-dimensional MoS2 nanosheet was prepared by a chemical exfoliation method and served as an excellent matrix for the detection of small molecules by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In comparison with organic matrices (CHCA, 3-AQ) and a graphene matrix, we found that a MoS2 matrix showed better performance in analysis of amino acids, peptides, fatty acids, and sulfonamides. A systematic comparison of the MoS2 matrix with both ion modes showed that mass spectra produced in negative ion mode featured a corresponding single deprotonated ion, which was rather different from the complex multiple alkali metal addition peaks present in positive ion mode. In addition, better sensitivity and reproducibility were obtained in negative ion mode. The ionization mechanism of MoS2 as a matrix in negative ion mode was further discussed. The deproton peak intensity of the analyte fatty acid decreased after the addition of the hole-scavenger KSCN, indicating that the ionization of the fatty acid was caused by the Auger complex effect of MoS2 and electron injection. Experiments have shown that the MoS2 matrix detects small molecules with good repeatability and can perform semiquantitative analysis of sulfonamides. Finally, the MoS2 matrix was employed for quantitative determination of sulfamethoxine in serum samples by an internal standard method. This MoS2-assisted laser desorption/ionization mass spectrometry (MoS2-assisted LDI MS) method provides a simple, rapid, high-throughput approach to evaluate the drug levels in the patient serum and can achieve convenient drug therapy monitoring.

On-line multi-residue analysis of fluoroquinolones and amantadine based on an integrated microfluidic chip coupled to triple quadrupole mass spectrometry.

An on-line multi-residue qualitative and quantitative analysis method for fluoroquinolones and amantadine using an integrated microfluidic chip was developed prior to directly coupling to triple quadrupole mass spectrometry (QQQ-MS). Six parallel channels consisting of sample filtration units and micro solid phase extraction (micro-SPE) columns were present in the specifically designed microfluidic device. Firstly, the impurities in the sample solution were trapped by the micropillars in the filtration units. The solution passed through the micro-SPE units packed with hydrophilic-lipophilic balanced (HLB) particles, and then the two classes of drugs were enriched. After washing, the targets were eluted and immediately electrosprayed for MS analysis. This approach allowed effective filtration, enrichment, elution, and MS detection without the introduction of an additional separation step after SPE. Direct electrospray ionization (ESI)-MS in multiple reaction monitoring (MRM) mode could not only ensure the high sensitivity of quantitative analysis, but also achieved accurate qualitative analysis towards targets using the MRM ratios, reducing the possibility of false positives. Good linear relationships were obtained by the internal standard (IS) method with a linear range of 1-200 ng mL-1 (R2 > 0.992). The mean recoveries of the eight target analytes were from 85.2% to 122% with the relative standard deviation (RSD) ranging from 5.6% to 20.3%. All this demonstrated that the developed microfluidic device could be a useful tool for rapid detection in the field of food safety.

Adaptive compressed sensing of Raman spectroscopic profiling data for discriminative tasks.

Raman spectroscopy is widely used in discriminative tasks. It provides a wide-range physio-chemical fingerprint in a rapid and non-invasive way. The Raman spectrometry uses a sensor array to convert photon signals into digital spectroscopic data. This analog-to-digital process can benefit from the compressed sensing (CS) technique. The major benefits include less memory usage, shorter acquisition time, and more cost-efficient sensor. Traditional compressed sensing and reconstruction is a series of mathematical operations performed on the signal. Meanwhile, for discriminative tasks, both the signal and the categorical information are involved. For such scenarios, this paper proposes a method that uses both domain signal and categorical information to optimize CS hyper-parameters, including 1) the sampling ratio or the sensing matrix, 2) the basis matrix for the sparse transform, and 3) the regularization rate or shrinkage factor for L1-norm minimization. A case study of formula milk brand identification proves the proposed method can generate effective compressed sensing while preserving enough discriminative power in the reconstructed signal. Under the optimized hyper-parameters, a 100% classification accuracy is retained by only sampling 20% of the original signal.

Highly Integrated Microfluidic Chip Coupled to Mass Spectrometry for Online Analysis of Residual Quinolones in Milk.

In this work, a highly integrated microfluidic chip with multifunction coupled to mass spectrometry (MS) was developed for online analysis of seven different regulated quinolones (QNs) in milk samples. Procedures of sample extraction, immunoaffinity enrichment, magnetic separation, and online elution were performed simultaneously on the specifically designed device. Based on the specificity of antibodies, direct (electrospray ionization) ESI-MS at full scan mode without liquid chromatography (LC) separation and further tandem mass spectrometry (MS/MS) analysis was developed for the identification of target QNs. One single isotope internal standard (IS) method was presented for quantitative analysis of seven QNs. Upon targeted online extraction and enrichment by antibody conjugated magnetic beads, seven QNs were quantitatively determined by the IS method with the linear range of 0.2/0.5-10 ng/mL (R2 > 0.991). The limits of detection (LODs) for the seven QNs were in the range of 0.047-0.490 ng/mL. This system permits automated on-chip immunoaffinity enrichment and accurate MS detection without additional off-line cleanup procedures.

Selection of aptamers against Lactoferrin based on silver enhanced and fluorescence-activated cell sorting.

We report a novel method for efficiently screening aptamers from a complex ssDNA library based on silver decahedral nanoparticles (AgNP) and fluorescence activated cell sorting (FACS). In this method, target protein (lactoferrin) and negative proteins (α-lactalbumin, ß-lactoglobulin, bovine serum albumin, casein) were respectively immobilized on polystyrene microspheres (PS) to form PSLac, PSα-Lac, PSß-Lac, PSBSA and PSCas. PSLac was firstly interacted with Cy5 labeled library (Lib), then hybridized with Cy5 modified silver decahedral nanoparticles (AgNPCy5) to form PSLac/Lib/AgNPCy5 conjugates. FACS was used to separate and collect PSLac/Lib/AgNPCy5 conjugates from complicated complex. AgNP was used to increase the fluorescence intensity in the selecting process and choose non-self-hybridization of Lib. Six aptamers (Ylac1, Ylac4, Ylac5, Ylac6, Ylac8 and Ylac9) were obtained after five-round of selection. These aptamers showed good specificity towards lactoferrin in the presence of negative proteins. The equilibrium dissociation constants (Kd) of six aptamers were calculated and all were in the nanomolar range. In a word, AgNP-FACS SELEX (AgFACS-SELEX) is a rapid, sensitive and highly efficient method for screening aptamers.

Magnetic nanoparticles and polydopamine amplified FP aptasensor for the highly sensitive detection of rHuEPO-α.

In this paper, an amplified fluorescence polarization (FP) aptasensor based on magnetic nanoparticles @polydopamine (MNP@PDA) was innovatively developed for sensitive detection of recombinant human erythropoietin-alpha(rHuEPO-α). The amplified FP signal was due to the large mass of protein and MNP@PDA. And this assay can be utilized for target separation or recycling based on the magnetic property of MNP@PDA through magnetic separation. Briefly, rHuEPO-α and MNP@PDA were added successively to react with the labeled aptamer (FAM-P1), which both contributed to the increase of FP signal via the formation of FAM-P1-rHuEPO-α and particularly FAM-P1-MNP@PDA complex. The strong interaction between MNP@PDA and FAM-P1 ensured the high efficiency of mass amplification and magnetic separation. As a result, the detection limit for rHuEPO-α was 0.12 pM, 4 orders of magnitude lower than original assay. Besides, three kinds of rHuEPO-α injections, NuPIAO, Epogen and ESPO were used to evaluate the selectivity of this assay in complex matrix with reasonable standard deviation. In a word, this work provides a simple, rapid, non-modified, highly sensitive and selective sensing platform for the detection of rHuEPO-α.

Multivalent aptasensor array and silver aggregated amplification for multiplex detection in microfluidic devices.

Herein, we developed a rapid and sensitive aptamers-based sandwich assay in microfluidic devices based on multivalent aptasensor array (MAA) chip and silver aggregated amplification (SAA) strategy for the detection of two biomarkers. Firstly, aptamers-modified silver nanoparticles were dotted in array to form MAA chip. Then PDMS was used to form a microfluidic device. After that, target proteins and two kinds of aptamer-modified silver nanoparticles (Tag-A and Tag-B) were rapidly injected into the microfluidic device. The aptamer on MAA chip recognized target, and the target also bound with Tag-A and Tag-B which could aggregate with each other to amplify fluorescence signal. Based on MAA chip and SAA strategy in microfluidic device, a linear response to PDGF-BB (r = 0.999) was obtained in the concentration range from 16 pg mL-1 to 250 ng mL-1, and the detection limit was 1.4 pg mL-1. In addition, a linear response to PDGF-BB (r = 0.992) was obtained in the concentration range from 16 pg mL-1 to 250 ng mL-1 in 10% blood serum with detection limit of 7.8 pg mL-1. Ultimately, this assay was used to simultaneously detect PDGF-BB and VEGF-165, and the results showed good specificity and sensitivity. This assay can also be expanded to sensitive and high-throughput detection of other protein biomarkers by coupling of various aptamers with nanoparticles.

Disposable MoS2-Arrayed MALDI MS Chip for High-Throughput and Rapid Quantification of Sulfonamides in Multiple Real Samples.

In this work, we demonstrate, for the first time, the development of a disposable MoS2-arrayed matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) chip combined with an immunoaffinity enrichment method for high-throughput, rapid, and simultaneous quantitation of multiple sulfonamides (SAs). The disposable MALDI MS chip was designed and fabricated by MoS2 array formation on a commercial indium tin oxide (ITO) glass slide. A series of SAs were analyzed, and clear deprotonated signals were obtained in negative-ion mode. Compared with MoS2-arrayed commercial steel plate, the prepared MALDI MS chip exhibited comparable LDI efficiency, providing a good alternative and disposable substrate for MALDI MS analysis. Furthermore, internal standard (IS) was previously deposited onto the MoS2 array to simplify the experimental process for MALDI MS quantitation. 96 sample spots could be analyzed within 10 min in one single chip to perform quantitative analysis, recovery studies, and real foodstuff detection. Upon targeted extraction and enrichment by antibody conjugated magnetic beads, five SAs were quantitatively determined by the IS-first method with the linear range of 0.5-10 ng/mL ( R2 > 0.990). Good recoveries and repeatability were obtained for spiked pork, egg, and milk samples. SAs in several real foodstuffs were successfully identified and quantified. The developed method may provide a promising tool for the routine analysis of antibiotic residues in real samples.

Aptamer-functionalized nano/micro-materials for clinical diagnosis: isolation, release and bioanalysis of circulating tumor cells.

Detection of rare circulating tumor cells (CTCs) in peripheral blood is a challenging, but necessary, task in order to diagnose early onset of metastatic cancer and to monitor treatment efficacy. Over the last decade, step-up produced aptamers have attracted great attention in clinical diagnosis. They have offered great promise for a broader range of cell-specific recognition and isolation. In particular, aptamer-functionalized magnetic particles for selective extraction of target CTCs have shown reduced damage to cells and relatively simple operation. Also, efforts to develop aptamer-functionalized microchannel/microstructures able to efficiently isolate target CTCs are continuing, and these efforts have brought more advanced geometrically designed substrates. Various aptamer-mediated cell release techniques are being developed to enable subsequent biological studies. This article reviews some of these advances in aptamer-functionalized nano/micro-materials for CTCs isolation and methods for releasing captured CTCs from aptamer-functionalized surfaces. Biological studies of CTCs after release are also discussed.

Silver enhanced ratiometric nanosensor based on two adjustable Fluorescence Resonance Energy Transfer modes for quantitative protein sensing.

We developed a silver decahedral nanoparticles (Ag10NPs)-enhanced ratiometric Fluorescence Resonance Energy Transfer (FRET) nanosensor based on two adjustable FRET modes. Alexa Fluor 488 (Alexa) and Cyanine3 (Cy3)-aptamer-Black hole quencher-2 (BHQ-2) were bound with Ag10NPs to form the ratiometric FRET nanosensor (Ag-Alexa/Cy3/BHQ-2). Alexa act as donor and Cy3 as acceptor in the FRET mode 1 while Cy3 was donor and BHQ-2 was acceptor in the FRET mode 2. In the absence of platelet-derived growth factor (PDGF-BB), the fluorescence intensity of Alexa was lowest while that of Cy3 was highest. Upon the addition of PDGF-BB, Cy3-aptamer-BHQ-2 binds with PDGF-BB resulting in the change of structure of aptamer. The fluorescence intensity of Alexa increased while that of Cy3 decreased. In addition, the fluorescence intensity ratio of Alexa to Cy3 increased remarkably with PDGF-BB concentration in the range of 0.4-400ng/mL. A good linear response was obtained when the PDGF-BB concentrations were in the range of 3.1-200ng/mL, with the limit of detection at 0.4ng/mL. When compared to sensors without Ag10NPs (Alexa/Cy3/BHQ-2) and one without BHQ-2 (Ag-Alexa/Cy3), the new nanosensor Ag-Alexa/Cy3/BHQ-2 showed remarkable increase in sensitivity.

MoS2/Ag nanohybrid: A novel matrix with synergistic effect for small molecule drugs analysis by negative-ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

This paper reports a facile synthesis of molybdenum disulfide nanosheets/silver nanoparticles (MoS2/Ag) hybrid and its use as an effective matrix in negative ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The nanohybrid exerts a strong synergistic effect, leading to high performance detection of small molecule analytes including amino acids, peptides, fatty acids and drugs. The enhancement of laser desorption/ionization (LDI) efficiency is largely attributed to the high surface roughness and large surface area for analyte adsorption, better dispersibility, increased thermal conductivity and enhanced UV energy absorption as compared to pure MoS2. Moreover, both Ag nanoparticles and the edge of the MoS2 layers function as deprotonation sites for proton capture, facilitating the charging process in negative ion mode and promoting formation of negative ions. As a result, the MoS2/Ag nanohybrid proves to be a highly attractive matrix in MALDI-TOF MS, with desired features such as high desorption/ionization efficiency, low fragmentation interference, high salt tolerance, and no sweet-spots for mass signal. These characteristic properties allowed for simultaneous analysis of eight different drugs and quantification of acetylsalicylic acid in the spiked human serum. This work demonstrates for the first time the fabrication and application of a novel MoS2/Ag hybrid, and provides a new platform for use in the rapid and high throughput analysis of small molecules by mass spectrometry.

Microfluidic chip-based silver nanoparticles aptasensor for colorimetric detection of thrombin.

In this paper, a colorimetric silver nanoparticles aptasensor (aptamer-AgNPs) was developed for simple and straightforward detection of protein in microfluidic chip. Surface-functionalized microfluidic channels were employed as the capture platform. Then the mixture of target protein and aptamer-AgNPs were injected into the microfluidic channels for colorimetric detection. To demonstrate the performance of this detection platform, thrombin was chosen as a model target protein. Introduction of thrombin could form a sandwich-type complex involving immobilized AgNPs. The amount of aptamer-AgNPs on the complex augmented along with the increase of the thrombin concentration causing different color change that can be analyzed both by naked eyes and a flatbed scanner. This method is featured with low sample consumption, simple processes of microfluidic platform and straightforward colorimetric detection with aptamer-AgNPs. Thrombin at concentrations as low as 20pM can be detected using this aptasensor without signal amplification. This work demonstrated that it had good selectivity over other proteins and it could be a useful strategy to detect other targets with two affinity binding sites for ligands as well.

Multifunctional aptamer-silver conjugates as theragnostic agents for specific cancer cell therapy and fluorescence-enhanced cell imaging.

We fabricated a multifunctional theragnostic agent Ag-Sgc8-FAM for apoptosis-based cancer therapy and fluorescence-enhanced cell imaging. For cancer therapy, aptamers Sgc8 and TDO5 acted as recognizing molecules to bind CCRF-CEM and Ramos cells specifically. It was found that aptamer-silver conjugates (Ag-Sgc8, Ag-TDO5) could be internalized into cells by receptor-mediated endocytosis, inducing specific apoptosis of CCRF-CEM and Ramos cells. The apoptosis of cells depended on the concentration of aptamer-silver conjugates, as well as the incubation time between cells and aptamer-silver conjugates. The apoptotic effects on CCRF-CEM and Ramos cells were different. Annexin V/PI staining, AO/PI staining, MTT assays and ROS (reactive oxygen species) detection demonstrated the specific apoptosis of CCRF-CEM and Ramos cells. For fluorescence-enhanced cell imaging, Ag-Sgc8-FAM was prepared. Compared to Sgc8-FAM molecules, Ag-Sgc8-FAM was an excellent imaging agent as numerous Sgc8-FAM molecules were enriched on the surface of AgNPs for multiple binding with CCRF-CEM cells and signal amplification. Moreover, AgNPs could increase the fluorescence intensity of FAM by metal-enhanced fluorescence (MEF) effect. Therefore, aptamer-silver conjugates can be potential theragnostic agents for inducing specific apoptosis of cells and achieving cells imaging in real time.

Aptamer-functionalized silver nanoparticles for scanometric detection of platelet-derived growth factor-BB.

In this work, we reported a scanometric assay system based on the aptamer-functionalized silver nanoparticles (apt-AgNPs) for detection of platelet-derived growth factor-BB (PDGF-BB) protein. The aptamer and ssDNA were bound with silver nanoparticles by self-assembly of sulfhydryl group at 5' end to form the apt-AgNPs probe. The apt-AgNPs probe can catalyze the reduction of metallic ions in color agent to generate metal deposition that can be captured both by human eyes and a flatbed scanner. Two different color agents, silver enhancer solution and color agent 1 (10 mM HAuCl4+2 mM hydroquinone) were used to develop silver and gold shell on the surface of AgNPs separately. The results demonstrated that the formation of Ag core-Au shell structure had some advantages especially in the low concentrations. The apt-AgNPs probe coupled with color agent 1 showed remarkable superiority in both sensitivity and detection limit compared to the apt-AuNPs system. The apt-AgNPs system also produced a wider linear range from 1.56 ng mL(-1) to 100 ng mL(-1) for PDGF-BB with the detection limit lower than 1.56 ng mL(-1). The present strategy was applied to the determination of PDGF-BB in 10% serum, and the results showed that it had good specificity in complex biological media.

Nanoparticle-catalyzed reductive bleaching for fabricating turn-off and enzyme-free amplified colorimetric bioassays.

Nanoparticle-catalyzed reductive bleaching reactions of colored substrates are emerging as a class of novel indicator reactions for fabricating enzyme-free amplified colorimetric biosensing (turn-off mode), which are exactly opposite to the commonly used oxidative coloring processes of colorless substrates in traditional enzyme-catalyzed amplified colorimetric bioassays (turn-on mode). In this work, a simple theoretical analysis shows that the sensitivity of this colorimetric bioassay can be improved by increasing the amplification factor (kcatΔt), or enhancing the binding affinity between analyte and receptor (Kd), or selecting the colored substrates with high extinction coefficients (ε). Based on this novel strategy, we have developed a turn-off and cost-effective amplified colorimetric thrombin aptasensor. This aptasensor made full use of sandwich binding of two affinity aptamers for increased specificity, magnetic particles for easy separation and enrichment, and gold nanoparticle (AuNP)-catalyzed reductive bleaching reaction to generate the amplified colorimetric signal. With 4-nitrophenol (4-NP) as the non-dye colored substrate, colorimetric bioassay of thrombin was achieved by the endpoint method with a detection limit of 91pM. In particular, when using methylene blue (MB) as the substrate, for the first time, a more convenient and efficient kinetic-based colorimetric thrombin bioassay was achieved without the steps of acidification termination and magnetic removal of particles, with a low detection limit of 10pM, which was superior to the majority of the existing colorimetric thrombin aptasensors. The proposed colorimetric protocol is expected to hold great promise in field analysis and point-of-care applications.