Enhancing the sequence coverage of nanodiamond-extracted early secretory proteins from the Mycobacterium tuberculosis complex.

The unambiguous identification of protein species requires high sequence coverage. In this study, we successfully improved the sequence coverage of early secretory 10 kDa cell filtrate protein (CFP-10) and 6 kDa early secretory antigenic target (ESAT-6) proteins from the Mycobacterium tuberculosis complex (MTC) in broth culture media with the use of the 4-chloro-α-cyanocinnamic acid (Cl-CCA) matrix. Conventional matrices, α-cyano-hydroxy-cinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB), were also used for comparison. After nanodiamond (ND) extraction, the sequence coverage of the CFP-10 protein was 87% when CHCA and DHB matrices were used, and the ESAT-6 protein was not detected. On the other hand, the sequence coverage for ND-extracted CFP-10 and ESAT-6 could reach 94% and 100%, respectively, when the Cl-CCA matrix was used and with the removal of interference from bovine serum albumin (BSA) protein and α-crystallin (ACR) protein. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was also adopted to analyze the protein mass spectra. A total of 6 prominent ion signals were observed, including ESAT-6 protein peaks at mass-to-charge ratios (m/z) of ∼7931, ∼7974, ∼9768, and ∼9813 and CFP-10 protein peaks at m/z of ∼10 100 and ∼10 660. The ESAT-6 ion signals were always detected concurrently with CFP-10 ion signals, but CFP-10 ion signals could be detected alone without the ESAT-6 ion signals. Furthermore, the newly found ESAT-6 peaks were also confirmed using a Mag-Beads-Protein G kit with an ESAT-6 antibody to capture the ESAT-6 protein, which was also consistent with the sequence coverage analysis.

Real-Time Monitoring of the Evaporation and Fission of Electrospray-Ionized Polystyrene Beads and Bacterial Pellets at Elevated Temperatures.

This study uses real-time monitoring, at microsecond time scales, with a charge-sensing particle detector to investigate the evaporation and fission processes of methanol/micrometer-sized polystyrene beads (PS beads) droplets and bacterial particles droplets generated via electrospray ionization (ESI) under elevated temperatures. By incrementally raising capillary temperatures, the solvent, such as methanol on 0.75 µm PS beads, experiences partial evaporation. Further temperature increase induces fission, and methanol molecules continue to evaporate until PS ions are detected after this range. Similar partial evaporation is observed on 3 µm PS beads. However, the shorter period of the fission temperature range is necessary compared to 0.75 µm PS beads. For the spherical-shaped bacterium, Staphylococcus aureus, the desolvation process shows a similar fission period as compared to 0.75 µm PS beads. Comparably, the rod-shaped bacteria, Escherichia coli EC11303, and E. coli strain W have shorter fission periods than S. aureus. This research provides insights into the evaporation and fission mechanisms of ESI droplets containing different sizes and shapes of micrometer-sized particles, contributing to a better understanding of gaseous macroion formation.

Fluorescent nanodiamond immunosensors for clinical diagnostics of tuberculosis.

Fluorescent nanodiamonds (FNDs) are carbon nanoparticles containing a dense ensemble of nitrogen-vacancy defects as color centers. These centers have exceptional photostability and unique quantum properties, making them useful for ultrasensitive biosensing applications. This work employed FNDs conjugated with antibodies as magneto-optical immunosensors for tuberculosis (TB) diagnostics using competitive spin-enhanced lateral flow immunoassay (SELFIA). ESAT6 (6-kDa early secretory antigenic target) of Mycobacterium tuberculosis is a clinical marker of TB. We evaluated the assay's performance using the recombinant ESAT6 antigen and its antibodies noncovalently coated on FNDs. A detection limit of ∼0.02 ng mL-1 was achieved with the lateral flow membrane strip pre-structured with a narrow channel of 1 mm width. Adopting a cut-off value of 24.0 ng mm-1 for 100-nm FNDs on the strips, the method detected 49 out of 50 clinical samples with Mycobacterium tuberculosis complexes. In contrast, none of the assays for 10 clinical samples with non-tuberculous mycobacteria (NTM) isolates exhibited the presence of ESAT6. These results suggest that the SELFIA platform is applicable for TB detection and can differentiate TB from NTM infections, which also affect the human respiratory system. The FND-enabled immunosensing techniques are versatile and promising for early detection of TB and other diseases, opening a new avenue for biomedical applications of carbon-based nanomaterials.

Optoacoustic classification of diabetes mellitus with the synthetic impacts via optimized neural networks.

A highly accurate classification of diabetes mellitus (DM) with the synthetic impacts of several variables is first studied via optoacoustic technology in this work. For this purpose, an optoacoustic measurement apparatus of blood glucose is built, and the optoacoustic signals and peak-peak values for 625 cases of in vitro rabbit blood are obtained. The results show that although the single impact of five variables are obtained, the precise classification of DM is limited because of the synthetic impacts. Based on clinical standards, different levels of blood glucose corresponding to hypoglycaemia, normal, slight diabetes, moderate diabetes and severe diabetes are employed. Then, a wavelet neural network (WNN) is utilized to establish a classification model of DM severity. The classification accuracy is 94.4 % for the testing blood samples. To enhance the classification accuracy, particle swarm optimization (PSO) and quantum-behaved particle swarm optimization (QPSO) are successively utilized to optimize WNN, and accuracy is enhanced to 98.4 % and 100 %, respectively. It is demonstrated from comparison between several algorithms that optoacoustic technology united with the QPSO-optimized WNN algorithm can achieve precise classification of DM with synthetic impacts.

A review: The nutrition components, active substances and flavonoid accumulation of Tartary buckwheat sprouts and innovative physical technology for seeds germinating.

Compared with the common grain, Tartary buckwheat enjoys higher nutritional value. Some distinctive nutrition associated with physiological activity of Tartary buckwheat is valuable in medicine. In addition, it's a good feed crop. In the paper, the main components (starch, protein, amino acid, fatty acid and mineral) and polyphenol bioactive components in Tartary buckwheat and its sprouts were reviewed, and the accumulation of flavonoids in sprouts during germination, especially the methods, synthetic pathways and mechanisms of flavonoid accumulation was summarized. The research on bioactive components and health benefits of Tartary buckwheat also were reviewed. Besides, the applications of innovative physical technology including microwave, magnetic, electromagnetic, ultrasonic, and light were also mentioned and highlighted, which could promote the enrichment of some active substances during seeds germination and growth of Tartary buckwheat sprouts. It would give a good support and benefit for the research and processing of Tartary buckwheat and its sprouts in next day.

Qualitative and Quantitative Assessments of Apple Quality Using Vis Spectroscopy Combined with Improved Particle-Swarm-Optimized Neural Networks.

Exploring a cost-effective and high-accuracy optical detection method is of great significance in promoting fruit quality evaluation and grading sales. Apples are one of the most widely economic fruits, and a qualitative and quantitative assessment of apple quality based on soluble solid content (SSC) was investigated via visible (Vis) spectroscopy in this study. Six pretreatment methods and principal component analysis (PCA) were utilized to enhance the collected spectra. The qualitative assessment of apple SSC was performed using a back-propagation neural network (BPNN) combined with second-order derivative (SD) and Savitzky-Golay (SG) smoothing. The SD-SG-PCA-BPNN model's classification accuracy was 87.88%. To improve accuracy and convergence speed, a dynamic learning rate nonlinear decay (DLRND) strategy was coupled with the model. After that, particle swarm optimization (PSO) was employed to optimize the model. The classification accuracy was 100% for testing apples via the SD-SG-PCA-PSO-BPNN model combined with a Gaussian DLRND strategy. Then, quantitative assessments of apple SSC values were performed. The correlation coefficient (r) and root-square-mean error for prediction (RMSEP) in testing apples were 0.998 and 0.112 °Brix, surpassing a commercial fructose meter. The results demonstrate that Vis spectroscopy combined with the proposed synthetic model has significant value in qualitative and quantitative assessments of apple quality.

Development of a linear ion trap mass spectrometer capable of analyzing megadalton MALDI ions.

Detecting megadalton matrix-assisted laser desorption/ionization (MALDI) ions in an ion trap mass spectrometer is a technical challenge. In this study, megadalton protein and polymer ions were successfully measured by MALDI linear ion trap mass spectrometer (LIT-MS) for the first time. The LIT-MS is comprised of a Thermo linear ion trap mass analyzer and a highly sensitive charge-sensing particle detector (CSPD). A newly designed radio frequency (rf) scan mode with dipolar resonance ejection techniques is proposed to extend the mass range of LIT-MS up to one million Thomson (Th). We analyze high mass ions with mass-to charge (m/z) ratios ranging from 100 kTh to 1 MTh, including thyroglobulin, alpha-2-macroglobulin, immunoglobulins (e.g., IgG and IgM), and polymer (∼ 940 kTh) ions. Besides, it is also very challenging for ion trap mass spectrometry to detect megadalton ions at low concentrations. By adopting high affinity carboxylated/oxidized detonation nanodiamonds (oxDNDs) to enrich IgM molecules and form antibody-nanodiamond conjugates, we have successfully reached âˆ¼ 5 nM (5 µg/mL) concentration which is better than that by the other techniques.

Effects of microwave and exogenous l-phenylalanine treatment on phenolic constituents, antioxidant capacity and enzyme inhibitory activity of Tartary buckwheat sprouts.

The phenolic substances, antioxidant capacity, and enzyme inhibitory activity of germinated Fagopyrum tataricum (Tartary buckwheat) under different microwave and l-phenylalanine (l-Phe) were investigated for the potential of enriching polyphenols. With the germination of seeds, the contents of total phenolics and total flavonoids increased, the antioxidant capacity and enzyme inhibitory activity were enhanced. The highest contents of total phenolics and total flavonoids in Tartary buckwheat sprouts were 17.41 mg GAE/g and 6.26 g RE/100 g DW (7 days), respectively. Correlation analysis and principal component analysis indicated that T3 (microwave 250 W, 90 s; l-Phe 2.9 mmol/L) could effectively improve the content of polyphenols, enzyme inhibition activity and antioxidant capacity of Tartary buckwheat sprouts obviously. This study hopes to provide some new ideas for enriching phenolics and improving antioxidation of Tartary buckwheat sprouts.

Quantitative measurement of blood glucose influenced by multiple factors via photoacoustic technique combined with optimized wavelet neural networks.

In this work, the photoacoustic (PA) quantitative measurement of blood glucose concentration (BGC) influenced by multiple factors was firstly investigated. A set of PA detection system of blood glucose considering the comprehensive influence of five factors was established. The PA signals and peak-to-peak values (PPVs) of 625 rabbit whole blood were obtained under 625 influence combinations. Due to the accurate measurement of BGC limited by the overlap PA signals, wavelet neural network (WNN) was utilized to train the PPVs of blood glucose for 500 rabbit blood. The mean square error (MSE) of BGC for 125 testing blood was approximately 6.5782 mmol/L. To decrease the MSE, the parameters of WNN were optimized by particle swarm optimization (PSO), that is, PSO-WNN algorithm was employed. Under the optimal parameters, MSE of BGC was decreased to approximately 0.48005 mmol/L. To further improve the prediction accuracy of BGC, an improved nonlinear dynamic inertia weight (NDIW) strategy of PSO was proposed, and compared with other two kinds of dynamic inertia weight strategies. Under the optimal parameters, the MSE of BGC was decreased to approximately 0.2635 mmol/L. The comparison of nine algorithms demonstrate that the PA technique combined with PSO-WNN and the improved NDIW strategy is significant in the quantitative measurement of blood glucose influenced by multiple factors.

Effect of co-treatment of microwave and exogenous l-phenylalanine on the enrichment of flavonoids in Tartary buckwheat sprouts.

BACKGROUND: Tartary buckwheat is rich in flavonoids. The application of physical processing technology and exogenous materials treatment can effectively promote grain germination and the accumulation of bioactive secondary metabolites. The content of four flavonoids, the activities of key enzymes (phenylalanine ammonia-lyase (PAL), chalcone isomerase (CHI), flavonol synthase (FLS)) and the expression of key enzyme genes (FtPAL, FtCHI, FtFLS1, FtFLS2) in Tartary buckwheat sprouts treated with microwave and l-phenylalanine (l-Phe) were investigated, and the relationship between them was analyzed to explore the mechanism of promoting flavonoid accumulation, and to provide a theoretical basis for the development of functional Tartary buckwheat sprout food. RESULTS: Germination can promote the synthesis of flavonoids. The contents of chlorogenic acid and rutin in 7-day sprouts increased by 13 420.63% and 225.12% compared with seeds, respectively. Under the best treatment condition T3 (microwave 250 W, 90 s, 2.9 mmol L-1 L-Phe), the specific activities of PAL, CHI and FLS in 5-day-old sprouts increased by 47.84%, 53.04% and 28.02% compared with control check (CK), respectively; and the expression of FtPAL, FtCHI and FtFlS1 increased by 39.84%, 24.78% and 33.72% compared with CK, respectively. Correlation analysis showed that the content of flavonoids in Tartary buckwheat sprouts was significantly positively correlated with the specific activities of key enzymes (P < 0.01) and dynamically correlated with genes related to the synthesis of three enzymes. CONCLUSION: It suggested that microwave and l-Phe treatment may promote the synthesis of flavonoids by promoting the expression of key enzymes genes in phenylpropane metabolism and controlling the activity of key enzymes in phenylpropane metabolism. © 2022 Society of Chemical Industry.

Effects of Exogenous Caffeic Acid, L-Phenylalanine and NaCl Treatments on Main Active Components Content and In Vitro Digestion of Germinated Tartary Buckwheat.

Germination is an effective method for improving the nutritional value of Tartary buckwheat (TB). The effects of exogenous additive treatments (caffeic acid (CA), L-phenylalanine (L-Phe), NaCl) on germination, main active component contents and antioxidant activities before and after in vitro digestion of germinated TB were investigated. Compared with the natural growth group, the T4 group (CA 17 mg/L, L-Phe 2.7 mmol/L, NaCl 2.7 mmol/L) treatment increased the germination rate (67.50%), sprout length, reducing sugar (53.05%), total flavonoid (18.36%) and total phenolic (20.96%) content, and antioxidant capacity of TB. In addition, exogenous additives treatment induced the consumption of a lot of nutrients during seed germination, resulting in a decrease in the content of soluble protein and soluble sugar. The stress degree of natural germination on seeds was higher than that of low concentrations of exogenous additives, resulting in an increase in malondialdehyde content. In vitro digestion leads to a decrease in phenolics content and antioxidant capacity, which can be alleviated by exogenous treatment. The results showed that treatment with exogenous additives was a good method to increase the nutritional value of germinated TB, which provided a theoretical basis for screening suitable growth conditions for flavonoid enrichment.

Label-free quantitative proteomics reveals the mechanism of microwave-induced Tartary buckwheat germination and flavonoids enrichment.

The objective of this study was to reveal the mechanism underlying the effects of microwave treatment on Tartary buckwheat germination and flavonoids enrichment. Label-free quantitative proteomic analysis showed that Tartary buckwheat germinated at 3, 5, and 7 days after 300 W/50 s microwave treatment had 7, 5, and 5 differentially expressed proteins (DEPs) compared to those of control. These DEPs are mainly related to energy production and conversion, gene expression, and flavonoids metabolism. Based on KEGG analysis, the DEPs were mainly enriched in photosynthesis, RNA polymerase, flavonoid biosynthesis, phenylalanine metabolism, and phenylpropanoid biosynthesis metabolic pathways. Further, the upregulation of phenylalanine ammonia-lyase and flavonol synthase protein enzymes promoted germination and flavonoids accumulation in Tartary buckwheat. These findings reveal the mechanism of Tartary buckwheat germination and the enrichment of flavonoids induced by microwaves and provide a scientific basis for the development of functional foods for Tartary buckwheat.

Rapid Quantification of Polyhydroxybutyrate Polymer from Single Bacterial Cells with Mass Spectrometry.

Polyhydroxyalkanoate (PHA) is one of the biocompatible and biodegradable plastics that can be produced and accumulated as granules inside microorganisms. In this study, a new approach to rapidly quantify a short-chain-length PHA, polyhydroxybutyrate (PHB), produced from genetically engineered Escherichia coli containing phaCAB is presented. The mass of each bacterial cell was measured using a laser-induced radio frequency (rf) plasma charge detection quadrupole ion trap mass spectrometer (LIRFP CD QIT-MS), and then, the PHB contents were determined by calculating the change in cellular mass. The quantitative results showed that the PHB contents measured by LIRFP CD QIT-MS were consistent with those by reference analysis, gas chromatography (GC). The PHB content of each bacterial sample can be obtained within 20 min from sampling using LIRFP CD QIT-MS while GC analysis takes 2 days. In addition, LIRFP CD QIT-MS does not use any hazardous chemicals in cellular mass quantification as compared to GC. This indicates that LIRFP CD QIT-MS has potential in routine monitoring of PHB production.

Effects of Microwave Treatment on Structure, Functional Properties and Antioxidant Activities of Germinated Tartary Buckwheat Protein.

Tartary buckwheat protein (TBP) has promise as a potential source of novel natural nutrient plant protein ingredients. The modulating effects of microwave pretreatment at varying powers and times on the structure, functional properties, and antioxidant activities of germinated TBP were investigated. Compared with native germinated TBP, after microwave pretreatment, the content of free sulfhydryl groups in the germinated TBP increased, and the secondary structure changes showed a significant decrease in α-helix and an increase in random coil contents, and the intensity of the ultraviolet absorption peak increased (p < 0.05). In addition, microwave pretreatment significantly improved the solubility (24.37%), water-holding capacity (38.95%), emulsifying activity index (17.21%), emulsifying stability index (11.22%), foaming capacity (71.43%), and foaming stability (33.60%) of germinated TBP (p < 0.05), and the in vitro protein digestibility (5.56%) and antioxidant activities (DPPH (32.35%), ABTS (41.95%), and FRAP (41.46%)) of germinated TBP have also been improved. Among different treatment levels, a microwave level of 300 W/50 s gave the best results for the studied parameters. Specifically, microwave pretreatment could be a promising approach for modulating other germinated plant protein resources, as well as expanding the application of TBP.

Photoacoustic identification of blood authenticity based on quantum-behaved particle swarm optimized wavelet neural network.

To accurately identify the blood authenticity, a set of photoacoustic detection system was established. In experiments, five kinds of blood in total of 125 groups were used, the time-resolved photoacoustic signals and peak-to-peak spectra were obtained in 700 to 1064 nm. Experimental results showed the accurate identification of blood authenticity was limited due to overlap of signals and spectra. To solve the problem, wavelet neural network (WNN) was employed to supervised train peak-to-peak spectra of 100 samples. The correct rate was 72% for 25 test samples. To improve correct rate, the parameters of WNN were optimized by quantum-behaved particle swarm optimization (QPSO) algorithm. Meanwhile, the effects of neurons number, learning rate factors, iteration times and training times on correct rate were studied and compared with WNN and WNN-PSO algorithms. Results showed the correct rate of WNN-QPSO was increased to 96%. Then, three kinds of dynamic contraction-expansion coefficients were used. Under the optimal dynamic coefficient, the correct rate reached 100%. Moreover, the truncated mean stabilization strategy (TMSS) was coupled to improve the convergent speed. Finally, 10 algorithms were compared. Results demonstrated that photoacoustic spectroscopy combined with WNN-QPSO coupled with TMSS and dynamic contraction-expansion coefficient had an excellent performance in the identification of blood authenticity.

Comparison of anesthesia effects between closed-loop and open-loop target controlled infusion of propofol using the bispectral index in abdominal surgery. / BISæŒ‡å¯¼ä¸‹å¼‚ä¸™é šé—­çŽ¯ä¸Žå¼€çŽ¯é¶æŽ§è¾“æ³¨è ¹éƒ¨æ‰‹æœ¯éº»é†‰æ•ˆæžœçš„æ¯”è¾ƒ.

OBJECTIVES: Bispectral index (BIS) can reflect the depth of propofol sedation. This study aims to compare the anesthetic satisfaction, anesthetic dose, and hemodynamic changes between closed-loop target controlled infusion (CLTCI) and open-loop target controlled infusion (OLTCI) during abdominal surgery. METHODS: From December 2016 to December 2018, 70 patients undergoing abdominal surgery under general anesthesia were selected in Beijing Hospital, including 51 males and 19 females, at the age from 49 to 65 years old. They were classified as grade I-II by the American Society of anesthesiologists (ASA) and were randomly divided into the CLTCI group and the OLTCI group (n=35). The CLTCI group received propofol CLTCI, and the target BIS value was set between 45 to 55; in the OLTCI group, the plasma target concentration was adjusted to maintain the BIS value between 40 to 60. Both groups were given remifentanil by target controlled infusion, and the plasma target concentration was 6.0 ng/mL. The percentages of adequate anesthesia time, deep anesthesia time, and light anesthesia time were calculated. The total induction dose of propofol, continuous infusion dose of propofol, predicted target propofol concentration of effect chamber, and continuous infusion dose of remifentanil were calculated. The times of automatic adjustment of propofol concentration in closed-loop system and manual adjustment of propofol concentration in the OLTCI group were recorded. Hemodynamic indexes were recorded, and the percentage of target mean arterial pressure (MAP) maintenance time and target heart rate (HR) maintenance time were calculated. Anesthesia time, operation time, recovery time, and extubation time were compared between the 2 groups. RESULTS: In the induction stage, the percentage of adequate anesthesia time in the CLTCI group was higher than that in the OLTCI group, and the percentage of deep anesthesia time in the CLTCI group was significantly lower than that in the OLTCI group (both P<0.05). In the maintenance stage, the percentage of light anesthesia time in the CLTCI group was significantly lower than that in the OLTCI group (P<0.05). The times of propofol adjustment in the CLTCI group was significantly more than that in the OLTCI group (P<0.001). The total induction dose of propofol in the CLTCI group was less than that in the OLTCI group (P<0.05), but there were no significant differences in the continuous infusion dose of propofol, predicted target concentration of propofol, continuous infusion dose of remifentanil between the 2 groups (all P>0.05). There were no significant differences in the percentages of target MAP maintenance time and target HR maintenance time between the 2 groups (both P>0.05). There was no intraoperative awareness in both groups, and there were no significant differences in operation time and anesthesia time between the 2 groups (both P>0.05). CONCLUSIONS: Compared with propofol OLTCI, anesthesia with propofol CLTCI under BIS guidance can maintain a more appropriate depth of anesthesia sedation and more stable hemodynamics.

Case report: life-threatening coronary artery spasm under transversus abdominis plane block in combination with general anesthesia.

BACKGROUND: Many cases of coronary artery spasm (CAS) during general plus epidural anesthesia have been reported. But transversus abdominis plane(TAP) block in combination with general anesthesia has not been reported to be a cause of CAS, let alone a life-threatening CAS. CASE PRESENTATION: In this case report, we present a case of a patient with CAS accompanied by ventricular fibrillation under general anesthesia with TAP block. CONCLUSION: Coronary artery spasm, even life-threatening CAS, may occur during TAP block in combination with general anesthesia.

Ionization of Submicrometer-Sized Particles by Laser-Induced Radiofrequency Plasma for Mass Spectrometric Analysis.

A laser-induced rf plasma (LIRFP) ion source was developed to ionize submicrometer-sized particles for the first time. The LIRFP ion source can increase the charge of those particles to several thousand charges via charge exchange reactions so that those particles can be trapped and analyzed with a charge detection quadrupole ion trap-mass spectrometer (CD QIT-MS). Different reagent gases for charge exchange reaction were investigated, viz. argon, nitrogen, oxygen, methane, helium, krypton, xenon, argon/methane (with ratios of 10:1 and 2:1), argon/nitrogen (with a ratio of 1:1), nitrogen/oxygen (10:1), krypton/methane (10:1), and air. The average charge of 0.75 µm polystyrene particles could reach 1631 using an argon/methane mixture with a ratio of ∼10:1. The average charges for freeze-dried Escherichia coli EC11303, Escherichia coli strain W, and Staphylococcus aureus were 842, 1112, and 971, respectively, with a mass-to-charge ratio ( m/ z) range from 107 to 108; and the average masses were 3.5 × 1010 Da, 6.0 × 1010 Da, and 5.6 × 1010 Da, respectively. The average mass and charge of the vaccinia virus were ∼9.1 × 109 Da and ∼708 with a m/ z of ∼107. This LIRFP CD QIT-MS method was rapid with only 20 min for each sample measurement.

Forced dried droplet method for MALDI sample preparation.

A forced dried droplet method (FDD) is developed to overcome the drawbacks of the conventional dried-droplet (DD) method for matrix assisted laser desorption/ionization (MALDI) sample preparation. The crystals produced by the DD method are heterogeneous and irregularly distributed, and thus many methods have tried to solve the problems. However, most of them spend more time or need additional instruments to generate homogeneous microcrystals. The FDD sample preparation method can produce uniform microcrystals with homogeneous size distribution in few minutes without additional instruments. Stirring the sample spot solution (an agitation process) with a pipette tip can change the crystal size distribution which is observed by the microscope. Mass spectrometric analysis shows that the smaller the crystal size is, the better the ion signal intensity is. The formation of microcrystals can be explained with the effective rate of secondary nucleation. The relative standard deviation (RSD) of the FDD method is ∼16% which is comparable to the two-layer (TL) method and is better than the DD method.