Micropipette Tip Research Articles

Simple cost‐effective larval injection method for dsRNA delivery to induce RNAi response in Helicoverpa armigera (Hübner)

AbstractMicroinjection is considered as an effective method for dsRNA delivery in insects. It also facilitates the delivery of a precise quantity of dsRNA in the host insect, inducing an efficient RNAi response. However, the microinjection method needs prior optimization of several parameters like concentration of dsRNA, site of injection, developmental stage of insect etc. for inducing effective RNAi response. Moreover, sophisticated microinjection devices are largely expensive with high maintenance cost. The Old‐World bollworm, Helicoverpa armigera (Hübner) is known to be a detrimental polyphagous pest with widespread infestations across the globe. In the present study, we demonstrate a low‐cost and effective dsRNA delivery method for inducing RNAi response in H. armigera with the aid of basic insulin injection syringe and fabricated micropipette tip. In order to validate the RNAi response following dsRNA injection, we have selected three key genes from the chitin biosynthesis pathway of the insect. Besides these, argonaute 1 (ago1) was also used as an indicator gene for dsRNA‐mediated RNAi induction. Delivery of dsRNA using injection with insulin syringe caused significant upregulation of the ago1 gene in the insect irrespective of any of the three target genes concerned viz. HaNAGK (3.9 fold; p < 0.001), HaAGM (6.3 fold; p < 0.001) and HaUAP (5.9 fold; p < 0.01) respectively, as compared to control injected with nuclease‐free water. The dsRNA‐injected insects showed aberrant developmental symptoms typical of impeded chitin synthesis, eventually leading to anomalous ecdysis with substantial mortality (up to 69.04%), as compared to controls. The described protocol reduces insect injury, enabling easy restraining of larva and quick execution of dsRNA injection with efficient RNAi response.

Disposable-micropipette tip supported electrified liquid-organogel interface as a platform for sensing acetylcholine.

Sensing acetylcholine has been predominantly based on enzymatic strategies using acetylcholine esterase and choline oxidase because of its electrochemical inertness. Electrified liquid-liquid interfaces are not limited to oxidation/reduction processes, and can be utilized to detect non-redox molecules which cannot be detected using conventional solid electrodes. In this study, a disposable micropipette tip based liquid-organogel interface, in the presence/absence of calixarene has been developed as a platform for sensing acetylcholine. We also explored a liquid-liquid interface approach for sensing acetylcholine using a pre-pulled glass micropipette. In both approaches, the configuration, i.e., liquid-organogel and liquid-liquid interface-current linearly increases during the backward transfer of acetylcholine. The simple and facilitated ion transfer of acetylcholine across the liquid-organogel exhibited a linear range of 10-50 μM and 1-30 μM with a detection limit of 0.18 μM and 0.23 μM and a sensitivity of 9.52 nA μM-1 and 9.20 nA μM-1, respectively. Whereas, the detection limit of simple and facilitated ion transfer of liquid-liquid interface using pre-pulled glass micropipette was found to be 0.42 μM and 0.13 μM with a sensitivity of 5 × 10-3 nA μM-1 and 3.39 × 10-2 nA μM-1. The results indicate that the liquid-organogel configuration supported on a disposable micropipette tip without any pre-fabrication is highly suitable for electrified soft interface sensing applications.

Electrosynthesis of Au nanocluster embedded conductive polymer films at soft interfaces using dithiafulvenyl-functionalized pyrene.

Nanoparticle (NP) embedded conductive polymer films are desirable platforms for electrocatalysis as well as biomedical and analytical applications. Increased catalytic and analytical performance is accompanied by concomitant decreases in NP size. Herein, highly reproducible electrogeneration of low dispersity Au nanocluster embedded ultra-thin (∼2 nm) conductive polymer films at a micro liquid|liquid interface is demonstrated. Confinement at a micropipette tip facilitates a heterogeneous electron transfer process across the interface between two immiscible electrolyte solutions (ITIES), between KAuCl4(aq) and a dithiafulvenyl-substituted pyrene monomer, 4,5-didecoxy-1,8-bis(dithiafulven-6-yl)pyrene (bis(DTF)pyrene), in oil, i.e., a w|o interface. At a large ITIES the reaction is spontaneous, rapid, and proceeds via transfer of AuCl4- to the oil phase, followed by homogeneous electron transfer generating uncontrolled polymer growth with larger (∼50 nm) Au nanoparticles (NPs). Thus, miniaturization facilitates external, potential control and limits the reaction pathway. Atomic (AFM) and Kelvin probe force microscopies (KPFM) imaged the topography and work function distribution of the as-prepared films. The latter was linked to nanocluster distribution.

Pharmaceutical Sorption to Lab Materials May Overestimate Rates of Removal in Lab-Scale Bioreactors.

Environmental contamination from pharmaceuticals has received increased attention from researchers in the past 20 years. As such, numerous lab-scale studies have sought to characterize the effects of these contaminants on various targets, as well as determine improved removal methods. Many studies have used lab-scale bioreactors to investigate pharmaceutical effects on wastewater bacteria, as wastewater treatment plants often act as reservoirs for pharmaceuticals. However, few-if any-of these studies report the specific lab materials used during testing, such as tubing or pipette tip type. In this study, the pharmaceuticals erythromycin, diclofenac, and gemfibrozil were exposed to different micropipette tips, syringe filters, and tubing types, and losses over time were evaluated. Losses to tubing and syringe filters were particularly significant and neared 100%, depending on the pharmaceutical compound and length of exposure time. Results discussed herein indicate that pharmaceutical sorption to various lab supplies results in decreases to both dosed and quantified pharmaceutical concentrations. Studies that fail to consider this source of loss may therefore draw inaccurate conclusions about pharmaceutical effects or removal efficiencies.

Heroin detection in a droplet hosted in a 3D printed support at the miniaturized electrified liquid-liquid interface

Simple sensing protocols for the detection of illicit drugs are needed. Electrochemical sensing is especially attractive in this respect, as its cost together with the analytical accuracy aspires to replace still frequently used colorimetric tests. In this work, we have shown that the interfacial transfer of protonated heroin can be followed at the electrified water-1,2-dichloroethane interface. We have comprehensively studied the interfacial behavior of heroin alone and in the presence of its major and abundant cutting agents, caffeine and paracetamol. To maximally increase developed sensing protocol applicability we have designed and 3D printed a platform requiring only a few microliters of the aqueous and the organic phase. The proposed sensing platform was equipped with a cavity hosting a short section of Ag/AgCl electrode, up to 20 µL of the aqueous phase and the end of the micropipette tip being used as a casing of a fused silica capillary having 25 µm as the internal pore diameter. The volume of the organic phase was equal to around 5 µL and was present inside the micropipette tip. We have shown that under optimized conditions heroin can be detected in the presence of caffeine and paracetamol existing in a sample with 10,000 times excess over the analyte of interest. The calculated limit of detection equal to 1.3 µM, linear dynamic range spanning to at least 50 µM, good reproducibility, and very low volume of needed sample is fully in line with forensic demands.

Microfluidic wound scratching platform based on an untethered microrobot with magnetic actuation

Cell migration is closely associated with various pathological conditions such as tumor invasion and metastasis as well as cell proliferation. The in vitro wound healing assay involves a simple and affordable technique and is used to examine the migration abilities of cells in a wound area created on confluent cell culture. Although this technique is easy to implement, it is not suitable for microfluidic chips. Microfluidic systems offer advantages such as examining the microenvironment of the cells and performing an analysis closer to the living system in a continuous flow setting. However, they are not compatible with the classical wound healing assay since they are closed systems, and their small channels are not suitable for wound creating via a micropipette tip. This work presents a functional system, which can be used to create wounds in a microfluidic platform without causing any negative effects on the cells and requiring the use of any chemicals. In this system, an untethered magnetic microrobot, which can be manipulated in 4 degrees of freedom (DOF) to create wounds with uniform sizes and different shapes within a closed microfluidic system was used. In addition, the effect of different wound geometries on wound healing was investigated. According to the results, triangle-shaped wound healed the slowest, while the plus-shaped wound healed the fastest. This study could open new lanes in the use of microrobots in lab-on-a-chip devices and can be extended to 3D cell cultures in the near future.

Nano-gold mediated aptasensor for colorimetric monitoring of acrylamide: Smartphone readout strategy for on-site food control

A simple aptasensor is embedded in the internal surface of a micropipette tip as the aptasensor substrate for the label-free monitoring of acrylamide. The aptasensor is based on the formation of the triple-helix molecular switch (THMS) structure of the DNA strands that induces the salt-enforced aggregation of gold nanoparticles (AuNPs). A smartphone imaging readout-based strategy is applied to quantify acrylamide. The developed aptasensor is novel for the naked-eye monitoring of the target through the color change of the solution inside the micropipette tip. The colorimetric aptasensor detects acrylamide in the concentration range of 0.05–200 nmol L−1 and at the trace level of 0.038 nmol L−1 with the comparable selectivity. The aptasensor can successfully quantify acrylamide in the chips, coffee, and bread samples with recoveries range from 92 to 102 %. The designed aptasensor paves an efficient device for the portable, on-site, facile, and real-time target sensing, superior for food safety control.

Use of solid phase microextraction in tips (SPME-Tips) for analysis of cocaine and biotransformation products in oral fluid by liquid chromatography tandem mass spectrometry (LC-MS/MS)

To develop and validate a method for analyzing cocaine and its metabolites using SPME-Tips and LC-MS/MS. Among the biological matrices used for analytical toxicology, oral fluid has advantages such as simplicity and non-invasive collection, the difficulty of adulteration, the low level of contamination risk and the possibility of recent abuse evaluation. Recently, nearly two million users have used cocaine in the US/Europe. When abused, cocaine is rapidly metabolized, mainly into methylecgonine and benzoylecgonine. Cocaethylene is formed in the concomitant use of cocaine with ethanol, and anhydroecgonine methyl ester (AEME) (in the crack abuse). Currently, in (green) analytical toxicology there is a growing interest in the use of new sample preparation techniques that are fast, efficient, automatable, and ecologically correct. Thus, methods based on microextraction processes have been developed, such as solid phase microextraction (SPME), a miniaturization of solid phase extraction. A variation of SPME technique was recently developed, consisting of a microfiber coated with extractor phase installed in a micropipette tip. In SPME-Tips, the extraction of analytes occurs in a extractor phase, allowing the direct immersion of the tip in biological fluids, causing the analytes to be adsorbed on the fiber and allowing the desorption to be carried out using small volume of more polar and less toxic solvents. The procedure was optimized, studying parameters such as salting-out effect, extraction time, sample agitation speed, elution solvent volume, and constitution. For extraction, SPME-Tips were immersed in 150 μL of oral fluid (collected with Quantisal) and kept under agitation at 500 rpm for 80 min. For the elution, 125 μL of methanolic solution with 0.1% formic acid was used, kept under agitation at 500 rpm for 30 min. The elution content was taken to LC-MS/MS, in which 5 μL were analyzed. The analyses were performed in a Nexera-UHPLC chromatographic system, coupled with a LCMS8060 mass spectrometer. The chromatographic separation was performed with Atlantis-T3 column (150 × 3.0 mm, 3.0 μm), maintained at 40 °C. The mobile phase consisted of ultra-pure water (A) and methanol (B), both containing formic acid (0.1%) and 2 mmol/L ammonium formate. The flow rate was set to 0.4 mL/min, and the elution gradient used started with 10%B for 1 min, followed by a linear increase to 100%B for 2 min and then maintained at 100%B for 2 min and returning to the proportion start in 0.1 min. This condition was maintained for another 3 min, completing a total of 8 min of chromatographic run. With this optimization, the method validation began, as recommended by the AAFS Standard Practices for Method Validation in Forensic Toxicology. For cocaine, benzoylecgonine, and cocaethylene, the correlation coefficient r 2 was greater than 0.98 (50 to 500 ng/mL). Good accuracy and precision results were obtained for low, medium and high QC. In this way, low extraction efficiency was observed to AEME and methylecgonine in C18 SPME-Tips. For the other analytes, the precision and accuracy values were in accordance to the criteria of the method validation guideline. Also, to prove the applicability of the method in real situations, 10 samples of oral fluid collected from individuals who participated in university parties will be analyzed. The samples were already collected and are stored in the Laboratory. A green analytical toxicology method was developed using SPME-Tips for the analysis of cocaine and its metabolites in oral fluid.

Automated Denudation of Oocytes.

Denudation is a technique for removal of the cumulus cell mass from oocytes in clinical intracytoplasmic sperm injection (ICSI). Manual oocyte denudation requires long training hours and stringent skills, but still suffers from low yield rate and denudation efficiency due to human fatigue and skill variations across operators. To address these limitations, this paper reports a robotic system for automated oocyte denudation. In this system, several key techniques are proposed, including a vision-based contact detection method for measuring the relative z position between the micropipette tip and the dish substrate, recognition of oocytes and the surrounding cumulus cells, automated calibration algorithm for eliminating the misalignment angle, and automated control of the flow rate based on the model of oocyte dynamics during micropipette aspiration and deposition. Experiments on mouse oocytes demonstrated that the robotic denudation system achieved a high yield rate of 97.0 ± 2.8% and denudation efficiency of 95.0 ± 0.8%. Additionally, oocytes denuded by the robotic system showed comparable fertilization rate and developmental competence compared with manual denudation. Our robotic denudation system represents one step towards the automation and standardization of ICSI procedures.

Clumps of mesenchymal stem cells/extracellular matrix complexes directly reconstruct the functional periodontal tissue in a rat periodontal defect model.

Periodontitis is an inflammatory disease characterized by tooth-supporting periodontal tissue destruction, including the cementum, periodontal ligament, and alveolar bone. To regenerate the damaged periodontal tissue, mesenchymal stem cells (MSCs) have attracted much scientific and medical attention. Recently, we generated clumps of MSCs/extracellular matrix (ECM) complexes (C-MSCs), which consist of cells and self-produced ECM. C-MSCs can be transplanted into lesion areas without artificial scaffold to induce tissue regeneration. To develop reliable scaffold-free periodontal tissue regenerative cell therapy by C-MSCs, this study investigated the periodontal tissue regenerative capacity of C-MSCs and the behavior of the transplanted cells. Rat bone marrow-derived MSCs were isolated from rat femur. Confluent cells were scratched using a micropipette tip and then torn off. The sheet was rolled to make a three-dimensional round clump of cells, C-MSCs. Then, ten C-MSCs were grafted into a rat periodontal fenestration defect model. To trace the grafted cells in the defect, PKH26-labeled cells were also employed. Micro-CT and histological analyses demonstrated that transplantation of C-MSCs induced successful periodontal tissue regeneration in the rat periodontal defect model. Interestingly, the majority of the cells in the reconstructed tissue, including cementum, periodontal ligaments, and alveolar bone, were PKH26 positive donor cells, suggesting the direct tissue formation by MSCs. This study demonstrates a promising scaffold-free MSCs transplantation strategy for periodontal disease using C-MSCs and offers the significance of multipotency of MSCs to induce successful periodontal tissue regeneration.

PCR-tips for rapid diagnosis of bacterial pathogens.

Micropipette tips are currently among the most used disposable devices in bioresearch and development laboratories. Their main application is the fractionation of solutions. New functionalities have recently been added to this device, widening their applications. This paper analyzed disposable micropipette tips as reagent holders of PCR reagents. PCR has become a prevalent and often indispensable technique in biological laboratories for various applications, such as the detection of coronavirus and other infectious diseases. A functional micropipette tip was implemented to simplify PCR analysis and reduce the contamination chances of deoxynucleotides and specific primers. This disposable device is prepared by tip coating processes of reagents, using polyvinyl alcohol polymer and additives. The coated layer is optimized to load and release PCR reagents efficiently. As a proof of concept, we show that the detection of Bordetella pertussis, the etiological agent of whooping cough whose diagnostic relies on PCR, can be quickly done using practical-functional tips. This device is an excellent example of testing the functionality and contribution of molecular diagnostic PCR tips.Key points • Functional micropipette tips are prepared by coating with dNTPs and primers. • Functional tips are used to replace dNTPs and primers in the PCR master mix. • PCR diagnostic of Bordetella pertussis is performed using functional tips. Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00253-022-12069-9.

Study on Ammonia Content and Distribution in the Microenvironment Based on Covalent Organic Framework Nanochannels.

A crack-free micrometer-sized compact structure of 1,3,5-tris(4-aminophenyl)benzene-terephthaldehyde-covalent organic frameworks (TAPB-PDA-COFs) was constructed in situ at the tip of a theta micropipette (TMP). The COF-covered theta micropipette (CTP) then created a stable liquid-gas interface inside COF nanochannels, which was utilized to electrochemically analyze the content and distribution of ammonia gas in the microenvironments. The TMP-based electrochemical ammonia sensor (TEAS) shows a high sensing response, with current increasing linearly from 0 to 50,000 ppm ammonia, owing to the absorption of ammonia gas in the solvent meniscus that connects both barrels of the TEAS. The TEAS also exhibits a short response and recovery time of 5 ± 2 s and 6 ± 2 s, respectively. This response of the ammonia sensor is remarkably stable and repeatable, with a relative standard deviation of 6% for 500 ppm ammonia gas dispensing with humidity control. Due to its fast, reproducible, and stable response to ammonia gas, the TEAS was also utilized as a scanning electrochemical microscopy (SECM) probe for imaging the distribution of ammonia gas in a microspace. This study unlocks new possibilities for using a TMP in designing microscale probes for gas sensing and imaging.

Xenotransplantation of cryopreserved human clumps of mesenchymal stem cells/extracellular matrix complexes pretreated with IFN-γ induces rat calvarial bone regeneration.

IntroductionThree-dimensional (3D) clumps of mesenchymal stem cells (MSCs)/extracellular matrix (ECM) complexes, composed with cells and self-produced intact ECM, can be grafted into defect areas without artificial scaffold to induce successful bone regeneration. Moreover, C-MSCs pretreated with IFN-γ (C-MSCsγ) increased the immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO) expression and thereby inhibited T cell activity. Xenotransplantation of human C-MSCsγ suppressed host T cell immune rejection and induced bone regeneration in mice. Besides, we have also reported that C-MSCs retain the 3D structure and bone regenerative property even after cryopreservation. To develop the “off-the-shelf” cell preparation for bone regenerative therapy that is promptly provided when needed, we investigated whether C-MSCsγ can retain the immunosuppressive and osteogenic properties after cryopreservation.MethodsConfluent human MSCs that had formed on the cellular sheet were scratched using a micropipette tip and then torn off. The sheet was rolled to make a round clump of cells. The round cell clumps were incubated with a growth medium for 3 days, and then C-MSCs were obtained. To generate C-MSCsγ, after 2 days’ culture, C-MSCs were stimulated with 50 ng/ml of IFN-γ. Both C-MSCs and C-MSCsγ were cryopreserved for 2 days and then thawed to obtain Cryo-C-MSCs and Cryo-C-MSCsγ, respectively. The biological properties of those cell clumps were assessed in vitro. In addition, to test whether human Cryo-C-MSCsγ attenuates immune rejection to induce bone regeneration, a xenograft study using a rat calvarial defect was performed.ResultsBoth IFN-γ pretreatment and cryopreservation process did not affect the 3D structure and cell viability in all human cell clumps. Interestingly, Cryo-C-MSCsγ showed significantly increased IDO mRNA expression equivalent to C-MSCsγ. More importantly, xenotransplantation of human C-MSCsγ and Cryo-C-MSCsγ induced rat calvarial bone regeneration by suppressing rat T cells infiltration and the grafted human cells reduction in the grafted area. Finally, there were no human donor cells in the newly formed bone, implying that the bone reconstruction by C-MSCsγ and Cryo-C-MSCsγ can be due to indirect host osteogenesis.ConclusionThese findings implied that Cryo-C-MSCsγ can be a promising bone regenerative allograft therapy that can be certainly and promptly supplied on demand.

High-pressure nanoESI of highly conductive volatile and non-volatile buffer solutions from a large Taylor cone: Effect of spray current on charge state distribution

Direct ESI-MS of proteins in nonvolatile buffer requires the generation of very fine initial droplets to resolve the peak for meaningful analysis. This is usually achieved using nanoESI capillary with an inner diameter of ∼1 μm or smaller. Recently we reported the generation of “true nanoelectrospray” from a micropipette tip with a large inner diameter under the super-atmospheric pressure condition (J. Am. Soc. Mass Spectrom.2022, 33, 491–498). The flow rate in the nL/min regime can be reproducibly adjusted using high voltage with the spray current as the indicator for flow rate. Here two further works are done on the same ion source. First, the validation of the spray current-flow rate relationship has been extended to 0.5 MPa, and the achievable minimum spray current is found to reduce with the ion source pressure. Second, the ion source is applied to study the flow rate effect on the charge state distribution in the nanoflow regime for proteins and peptides in volatile (formic acid and ammonium acetate) and the nonvolatile (phosphate buffer saline (PBS) and 1 M Tris-HCl) buffers. For the nonvolatile buffers, clean nanoESI mass spectra are only obtained at a low flow rate of ∼1 nL/min. The average charge state of the tested proteins is primarily determined by the type of buffer. For acidic solutions, the charge state shifts to a higher value at a lower flow rate. For non-denaturing solutions, the average charge number remained relatively constant but the variation with the flow rate appeared to be analyte and buffer dependent.

Accurate delivery of chemicals to a small target using a pressure-driven valved micropipette.

We describe a new method for accurately and reproducibly delivering a minute amount of a chemical to a small target in an aqueous environment. Our method is based on a micropipette with a check valve at its tip that can be opened and closed on demand. We demonstrate that this device can produce a flux of 10-12l in a short pulse lasting less than 100 ms. The finite width of the pulse is due to molecular dispersion of the chemical, in this case, fluorescein. The chemical distribution near the micropipette tip is measured and compared with the results of a numerical integration assuming stokeslet flow. Our technique is of general utility and has applications in microbiology and neuroscience when a precise control of the spatiotemporal chemical distribution around a specimen is desired.

A versatile user-friendly electrochemical cell with three 3D-pen-printed electrodes in a tiny micropipette tip

This work describes a practical, portable and versatile electrochemical cell containing three-electrode integrated in a small disposable micropipette tip. The electrodes were manufactured using 3D pen and a commercial carbon black-polylactic acid filament and the tip-based cell was fabricated using alternative materials. The cell requires low sample volumes (10 μL) and may be operated with several commercial micropipettes (manual and electronic) that performs the aspiration of solution before or simultaneously to the electrochemical measurement, resulting in electroanalysis under static or flowing conditions, respectively. As proof-of-concept, measurements of catechol were performed in stationary and hydrodynamic mode, in which the sensitivity of the hydrodynamic was around five-fold greater when compared to the stationary method (47 vs 9 nA μmol−1 L), due to the faster mass transport of the analyte. Moreover, the proposed cell exhibited analytical parameters for catechol comparable with a commercial screen-printed electrode. The versatility of the cell was also demonstrated for square wave anodic stripping voltammetry in the presence of lead ions, in which the deposition (pre-concentration) step was performed during the solution aspiration controlled by an electronic pipette. The obtained LOD was 6-fold better than a conventional system for ASV (0.010 vs 0.059 μmol L-1). The analytical feasibility of the cell was demonstrated through the determination of catechol and lead in tap and ground water, being obtained recoveries close to 100%. The proposed cell and is cheaper (< US$ 0.10), smaller and more versatile than other tip-based and 3D-printed cells, being promising for on-site analyses and education purposes.

Generation of Ions from Aqueous Taylor Cones near the Minimum Flow Rate: "True Nanoelectrospray" without Narrow Capillary.

Generating ultrafine charged droplets using electrospray is crucial for attaining high ionization efficiency for mass spectrometry. The size of the precursor charged droplets depends on the spray flow rate, and conventional wisdom holds that an electrospray of a nL/min flow rate (nanoelectrospray) is only possible using narrow capillaries with an inner diameter of ∼1 μm or smaller. Here, the electrospray of aqueous solutions with high electric conductivities generated from a large off-line capillary of 0.4 mm i.d. has been performed using a high-pressure ion source. The electric discharge is avoided by operating the ion source at 2.5 bar gauge pressure. The highly stable Taylor cone can be tuned to a near-hydrostatic state that exhibits the "true nanoelectrospray" properties, i.e., high salt tolerance and minimal ion suppression. The Q1/2 scaling law describing the electrospray current I and flow rate Q is found to be valid down to the nanoflow regime under a condition that is free of electric discharge. For a given solution, the flow rate and the size of the initial droplets and ionization species can be controlled with the spray current as the indicator for the instantaneous flow rate without changing the emitter capillary of different sizes. In regard to the application, the nanoelectrospray with a large micropipette tip is easy to use, free of clogging when dealing with viscous and high-salt buffer solutions, and with reduced surface interaction with the emitter inner surface. An acquisition of very clean mass spectra of proteins from concentrated solutions of nonvolatile salts such as phosphate-buffered saline is demonstrated.

Detection of multiply charged protein ions using matrix-assisted laser desorption/ionization mass spectrometry and a force-dried droplet method with a 2-nitrophloroglucinol matrix.

Conventional dried droplet (DD) methods show poor reproducibility in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) due to the frequent induction of a heterogeneous sample distribution. Recently, a forced dried droplet (FDD) sample preparation method was introduced to form homogeneous samples; this method improves the reproducibility of MALDI-MS analysis and generates highly multiply charged ions compared to DD methods. The FDD method utilizes secondary nucleation to generate a homogeneous sample distribution by applying an external force such as fluid shear stress by stirring the sample using a micropipette tip. In this study, a 2-nitrophloroglucinol (2-NPG) matrix was used for the DD and FDD sample preparation methods, and the charge state and homogeneity were compared by detecting multiply charged ions of proteins including cytochrome c, myoglobin, and immunoglobulin G (IgG) and measuring the relative standard deviation (RSD). FDD with a 2-NPG matrix produced a more homogeneous sample distribution and higher charge state ions than the DD method. FDD with a 2-NPG matrix was applied in MALDI-MS analysis of IgG fragments obtained from sequential reduction of IgG. In addition, FDD with intentional scratching of the MALDI plate by rotating a micropipette tip was found to provide similar or better reproducibility, higher charge state ions, and more uniformly distributed sample morphology compared to FDD without scratching.

Functionalization of Micropipette Tips with Hydrophobin-Laccase Chimera and Application to the Electrochemical Determination of Caffeic Acid in Tea Samples

Functionalization of Micropipette Tips with Hydrophobin-Laccase Chimera and Application to the Electrochemical Determination of Caffeic Acid in Tea Samples

Functionalization of Micropipette Tips with Hydrophobin-Laccase Chimera and Application to the Electrochemical Determination of Caffeic Acid in Tea Samples

Functionalization of Micropipette Tips with Hydrophobin-Laccase Chimera and Application to the Electrochemical Determination of Caffeic Acid in Tea Samples