Pipette Tip Research Articles

On reactive Ion Etching of Parylene-C with Simple Photoresist Mask for Fabrication of High Porosity Membranes to Capture Circulating and Exfoliated Tumor Cells

A high porosity micropore arrayed parylene membrane is a promising device that is used to capture circulating and exfoliated tumor cells (CTCs and ETCs) for liquid biopsy applications. However, its fabrication still requires either expensive equipment or an expensive process. Here, we report on the fabrication of high porosity (>40%) micropore arrayed parylene membranes through a simple reactive ion etching (RIE) that uses photoresist as the etching mask. Vertical sidewalls were observed in etched parylene pores despite the sloped photoresist mask sidewalls, which was found to be due to the simultaneous high DC-bias RIE induced photoresist melting and substrate pedestal formation. A theoretical model has been derived to illustrate the dependence of the maximum membrane thickness on the final pore-to-pore spacing, and it is consistent with the experimental data. A simple, yet accurate, low number (<50) cell counting method was demonstrated through counting cells directly inside a pipette tip under phase-contrast microscope. Membranes as thin as 3 μm showed utility for low number tumor cell capture, with an efficiency of 87–92%.

Abstract 221: Novel organotypic patient derived primary tumor tissues for oncology drug safety and efficacy studies

Abstract Organotypic, three-dimensional (3D) cancer tissue models (OCTM) have enabled investigators to develop predictive markers that can be used in preclinical cancer drug development. However, a drawback of the existing models is that they are mostly based on cell lines based are not physiological. In vitro development of 3D tumoroids using patient derived primary tumors (PDPT) in tumor microenvironment (TME) will have clinical significant and can predict human responses to cancer therapeutics. Here, we describe the generation of OCTM from patient-derived metastatic colorectal cancer, the second most deadly cancer. PDPT were grown in Matrigel for 5 passages and for biobanking purposes tumoroids were further expanded in faCellitate 384 well plates or Sun Bioscience plates to harvest large amounts of well-defined tumoroids cultured for over 21 days. Expanded tumoroids were collected and cryopreserved or gently mixed several times using a pipet tip to break up tumoroids into smaller fragments. These tumor fragments were mixed with endothelial cells, dendritic cells, fibroblasts and embedded in a collagen matrix to simulate TME. The collagen-cell mixture was then added to Transwells and induced to gel to form a collagen gel matrix that simulates TME. The tumoroids were cultured with a specialized medium that supports the growth of complex cell types. The tumor development was monitored daily using bright field microscopy and image analysis was used to monitor tumor size. To evaluate the utility of the OCTM, treatment was initiated at day 10 of the culture period by exposing tissues with the chemotherapeutic drug Cisplatin on days 0, 2, 4, 7, 9, and 11. After each treatment, OCTM tissues (N=2) were fixed for H&E and live/dead staining. Overall, the results of the study showed that: 1) PDPT can be expanded for 5 passages (this low passage number is not expected to change the phenotype of the tumor) and further expanded in high throughput format, 384 well plates, 2) the PDPT could be cultured with multiple cell types in a collagen-gel matrix to recreate the TME, 3) histological evaluation of the OCTM showed a glandular-like tumoroid, 4) immunohistochemical staining revealed that the PDPT in the in vivo-like microenvironment were positive for CK20, and 5) the PDPT shrank following 3 treatment cycles with Cisplatin (day 4). Conclusion: The data shows metastatic colorectal PDPT can be expanded in vitro and cultured in a complex tumor microenvironment on tissue culture inserts. These OCTMs can be used to screen drug safety and efficacy. This study opens new avenues for high-throughput cancer drug screening format using PDPT embedded in a in vivo like TME. This model can be used as a tool for personalized oncology drug testing and for translational assays tailored to other cancer types from our patient derived primary tumor pool. Citation Format: Seyoum Ayehunie, Megan Groves, Bryda Bryda, Alex Armento, Anthony Tolcher. Novel organotypic patient derived primary tumor tissues for oncology drug safety and efficacy studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 221.

The Application of Pipette-Tip and Magnetic Dummy-Template Molecularly Imprinted Solid-Phase Extraction Coupled with High-Performance Liquid Chromatography with Diode Array and Spectrofluorimetric Detection for the Determination of Coumarins in Cosmetic Samples

In this study, adsorbents based on molecularly imprinted polymers (MIPs) in two solid-phase extraction application forms, pipette tip and magnetic extraction, were used for the selective extraction of coumarins. The pipette-tip solid-phase extraction reduced solvent volumes; the magnetic MIP extraction was simple and effective for phase separation. Parameters affecting extraction, such as the amount of adsorbent, type of washing solvent, volume of the elution solvent, and extraction times for magnetic extraction, were optimized. The MIP-based adsorbents displayed high selectivity and extraction efficiency, resulting in recoveries ranging from 70.3 to 102.0% (RSD % less than 5.5%) for five coumarins under study, 6,7-dihydroxycoumarin-6-β-D-glucoside, coumarin, 7-methoxycoumarin, 6-methylcoumarin, and dicoumarol. The extracts were analyzed by high-performance liquid chromatography with diode array (DAD) and fluorescence (FLD) detectors, reaching limits of quantification of 0.5 and 0.9 µg·mL−1 for coumarin and dicoumarol detected by DAD and 0.001–0.012 µg·mL−1 for the other prohibited simple coumarins when used as a fragrance (detected by FLD). The proposed method was validated and its applicability was shown for the analysis of cosmetic samples like shower gel and perfume.

In vitro Wound Healing Effect of a Siddha Formulation: Gandhaga Thailam

In vitro Wound Healing Effect of a Siddha Formulation: Gandhaga Thailam

Automated wash and reuse of disposable pipette tips in a SARS-CoV-2 RT-qPCR diagnostic pipeline

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic led to global shortages in laboratory consumables, in particular for automated PCR. The Technical University of Denmark supported Danish hospitals from 2020 to 2022, conducting SARS-CoV-2 RT-qPCR on around 10,000 patient samples daily. We encountered shortages of disposable pipette tips used with automated liquid handlers that transferred oropharyngeal swab samples to 96-well microplates before RNA extraction. To enable tip reuse, we developed an automated protocol for washing tips with a 0.5 % sodium hypochlorite solution. This effectively eliminated carry-over of genomic material and the wash solution remained effective when stored in an open reservoir at ambient temperatures for 24 h. A three-day validation setup demonstrated the robustness of the tip wash protocol. Reducing the number of tips used for transferring samples to 96-well microplates from 96 to 8 enabled us to mitigate pipette tip shortages, lower costs, and minimize plastic waste generation.

“Halotolerant fungi secreting phytohormones and volatile organic compounds enhance growth and mineral content in finger millet under salinity stress”

Soil salinity is one of the major environmental stresses that significantly hampers crop yield worldwide. This study assessed the effectiveness of the rhizospheric fungus, Aspergillus sojae JUSPF-101, in enhancing the tolerance of finger millet seedlings to NaCl stress. The research also examined the role of JUSPF-101 in producing phytohormones and volatile organic compounds. The impact of A. sojae was evaluated on both primed and unprimed finger millet seedlings (using Petri dish and pipette tip bioassay methods) under NaCl stress conditions. A study was conducted in a greenhouse to confirm the growth characteristics and mineral composition of finger millet seedlings, both with and without inoculation of A. sojae JUSPF-101. These seedlings were exposed to varying concentrations of NaCl (0 and 400 mM).The current research demonstrated that A. sojae could withstand high concentrations of NaCl (up to 1200 mM) and high temperatures (45 °C). Using LC-MS/MS, fifteen phytohormones were identified in A. sojae grown under NaCl concentrations of 0 and 400 mM. These include IAA, SA, GA4, epibrasinolide, the trans isomer of zeatin, and cis-jasmone. The GC–MS analysis detected seventy-seven VOCs, with the major compound being 1-(4-Amino-furazan-3-yl)-5-propyl-1H-[1,2,3]triazole-4-carboxylic acid ethyl ester (8.7%). Moreover, finger millet seedlings that were not treated displayed a significant decrease in RWC and biochemical parameters such as proline, phenols, and flavonoids (p < 0.05), while there was an increase in MDA and H2O2 content. However, using A. sojae effectively alleviated the stress caused by NaCl and led to improvements in RWC, growth, and biochemical parameters.The application of A. sojae under 400 mM NaCl treatment, significantly increased morphological (shoot and root length) and biochemical parameters such as proline (1.67-fold), phenols (2.15-fold), and flavonoids (2.04-fold) while MDA (1.2-fold), and H2O2 (1.25-fold) decreased as compared to unprimed seedlings under NaCl stress. In a greenhouse environment, seedlings treated with A. sojae showed improved growth parameters and lower Na+/K+ and Na+/Ca2+ ratios. These findings suggest that applying A. sojae (JUSPF-101) could be an effective strategy for reducing NaCl stress in finger millet, contributing to sustainable food security.

A metal-organic framework signaling probe-mediated immunosensor for the economical and rapid determination of enrofloxacin in milk

Ensuring the safety of animal-derived foods requires the reliable and swift identification of enrofloxacin residues to monitor the presence of antibiotics. In this regard, we synthesized, tuned, and investigated the optical properties of a bimetallic metal–organic framework (Ce/Zr-UiO 66). The investigation was facilitated by employing a polydopamine-coated pipette tip with high adsorption efficiency, serving as an immunoreactive carrier. Subsequently, an immunofunctionalized variant of Ce/Zr-UiO 66, referred to as Ce/Zr-UiO 66@ Bovine serum albumin-enrofloxacin, was developed as an optical probe for the rapid and sensitive identification of enrofloxacin across a variety of samples. The method can accurately detect enrofloxacin at concentrations as low as 0.12 ng/mL, with a determination time of under 15 min; furthermore, it demonstrates exceptional efficacy when applied to food, environmental, and clinical samples. The implementation of this methodology offers a valuable means for cost-effective, rapid, and on-site enrofloxacin determination.

Bioinspired composite packed in blunt needles, integrated microextraction and determination of oxycodone and naloxone in saliva by substrate spray mass spectrometry

BackgroundOpioids are effective painkillers used for medical purposes. Their prolonged ingestion can provoke some side effects (including overdose or constipation) that are minimized by using opioid antagonists (e.g., naloxone). The rapid determination of opioids and their antagonists in biosamples is essential for an effective medical treatment. The direct combination of sample preparation and mass spectrometry (MS) fits well in this scenario. It can speed up the analysis achieving a good selectivity, which relies on the sample preparation and MS, and sensitivity levels. ResultsThis article presents a novel substrate-spray mass spectrometry interface based on a polydopamine-cotton (PDA-Cel) composite hosted inside the inner diameter of a 14-gauge blunt needle to determine oxycodone and naloxone in saliva samples. The needle is used as a microextraction device and a substrate for mass spectrometric analysis. The lack of sharpness of the 14-gauge (14G) blunt needles challenges the formation of the electrospray (ESI), and a commercial 10 μL pipette tip is proposed as a simple solution to this shortcoming. Under the optimum parameters, the proposed method was validated, obtaining limits of detection lower than 0.6 μg L−1, linear range up to 200 μg L−1, and linearity better than 0.9915. Relative standard deviation (RSD) and relative recoveries (RR) were studied at three different concentration levels (2, 40, and 200 μg L−1). RSD values were better than 20.7 %, and RR ranged from 90 to 114 %. Finally, a positive sample from a patient under medical treatment was analyzed. Significance and novelty14G blunt needles have been demonstrated as effective extraction devices due to their low price (<0.15 € per extraction unit), their better safety (avoiding finger pricking), and their higher hosting capacity (up to 8 mg of sorbent). The conductivity of stainless steel permits their use as electrospray emitters, making their direct combination to MS easier. The large variety of fibrous sorbents makes this approach versatile enough to be adapted to other analytical problems.

Rapid, high-throughput, cost-effective whole-genome sequencing of SARS-CoV-2 using a condensed library preparation of the Illumina DNA Prep kit.

Sequencing has played an invaluable role in the response to the COVID-19 pandemic. Ongoing work in this area, however, demands optimization of laboratory workflow to increase sequencing capacity, improve turnaround time, and reduce cost without compromising sequence quality. This report describes an optimized DNA library preparation method for improved whole-genome sequencing of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogen. The workflow advantages summarized here include significant time, cost, and consumable savings, which suggest that this new method is an efficient, scalable, and pragmatic alternative for SARS-CoV-2 whole-genome sequencing.

High throughput generating stable spheroids with tip-refill wafer.

Three-dimensional (3D) cell cultures have garnered significant attention in biomedical research due to their ability to mimic the in vivo cellular environment more accurately. The formation of 3D cell spheroids using hanging drops has emerged as a cost-effective and crucial method for generating uniformly-sized spheroids. This study aimed to validate the potential of a tip-refill wafer (TrW), a disposable laboratory item used to hold pipette tips, in facilitating 3D cell culture. The TrW allows for easy generation of hanging drops by pipetting the solution into the holes of the wafer. The mechanical stability of the hanging drops is ensured by the surface wettability and thickness of the TrW. Hanging drops containing 60-µL of solution remained securely attached to the TrW even when subjected to orbital shaking at 210rpm. The exceptional resistance to mechanical shaking enabled the use of inertial focusing to facilitate spheroid formation. This was demonstrated through live/dead cell staining, quantitative polymerase chain reaction (qPCR) analysis, and cytoskeleton staining, which revealed that horizontal orbiting at 60rpm for 15 min promoted cell aggregation and ultimately led to the formation of 3D spheroids. The spheroid harvest rate is 96.1%±3.5% across three TrWs, each containing 60 hanging drops. In addition to generating mono-culture 3D spheroids, the TrW-based hanging drop platform also enables the formation of multicellular spheroids, and on-demand pairing and fusion of spheroids. The TrW is a disposable item that does not require any fabrication or surface modification procedures, further enhancing its application potential in conventional biological laboratories.

An optimum study on the laser scanning confocal microscopy techniques for BiFC assay using plant protoplast

BackgroundThe bimolecular fluorescence complementation (BiFC) assay is commonly used for investigating protein–protein interactions. While several BiFC detection systems have been developed, there is a limited amount of research focused on using laser scanning confocal microscope (LSCM) techniques to observe protoplasts. Protoplasts are more susceptible to damage and instability compared to their original cell state due to the preparation treatments they undergo, which makes it challenging for researchers to manipulate them during observation under LSCMs. Therefore, it is crucial to utilize microscope techniques properly and efficiently in BiFC assays.ResultsWhen the target fluorescence is weak, the autofluorescence of chloroplast particles in protoplasts can interfere with the detection of BiFC signals localized in the nuclear region. Spectrum analysis revealed that chloroplast autofluorescence can be excited by lasers of various types, with the highest fluorescence signal observed at around 660 nm. Furthermore, our investigation into the impact of different pipette tips on the integrity of protoplast samples indicated that the utilization of cut tips with larger openings can mitigate cell breakage. We presented a workflow of LSCM techniques for investigating protoplast BiFC and discussed the microscopic manipulation involved in sample preparation and image capturing.ConclusionWhen the BiFC signals are weak, they may be affected by chloroplast autofluorescence. However, when used properly, the autofluorescence of chloroplasts can serve as an excellent internal marker for effectively distinguishing other signals. In combination with other findings, this study can provide valuable reference for researchers conducting BiFC assays and related studies.

Deep eutectic solvent-doped graphite as an efficient adsorbent for pipette tip micro-solid phase extraction of mitoxantrone from seawater, drug, urine and blood samples prior to its spectrophotometric determination

Deep eutectic solvent-doped graphite as an efficient adsorbent for pipette tip micro-solid phase extraction of mitoxantrone from seawater, drug, urine and blood samples prior to its spectrophotometric determination

Mir‐205‐5p as a regulator agent in cell migration processes

Aims/Purpose: Endothelial cell migration is enhanced in pathological processes of the eye involving high glucose, such as diabetic retinopathy (DR). The microRNA miR‐205‐5p is identified as an important regulator of vasculogenesis and could slow down cell migration. The aim of this study is to understand the role of mir‐205‐5p in endothelial cell proliferation under high glucose (HG) conditions.Methods: HUVEC were seeded at a density of 5.0 × 104 cells/cm2. After 48 h, a wound was produced by scratching cell monolayers with a pipette tip. The cells were treated with different conditioned mediums from ARPE‐19 cells: control media, control media and miR‐205‐5p mimic, HG media and HG media and miR‐205‐5p. Images were taken at 0, 4, 8 and 24 h. The gap distance was analysed with Image J (migration n = 4 and viability n = 6).Results: HUVEC cells treated with HG media from ARPE‐19 cells close the gap faster than HUVEC cells treated with ARPE‐19 control media or ARPE‐19 control media with mimic miR‐205‐5p. The administration of mimic miR‐205‐5p in HG media significantly reduce migration of HUVEC cells. Besides, the presence of HG for 5 days reduces ARPE‐19 cells viability however miR‐205‐5p mimic protect them, increasing cell viability in control.Conclusions: Angiogenesis is mostly due to an enhancement of endothelial cell migration. That seems to be related to a reduction of miR‐205‐5p, released from RPE cells. When a miR‐205‐5p mimic was added in the medium, cell migration slowed down.

Design and Validation of a Three-Dimensional Printer-Based System Enabling Rapid, Low-Cost Construction of the Biosensing Areas of Lateral Flow Devices for Immunoassays and Nucleic Acid Assays.

The COVID-19 pandemic proved the great usefulness of lateral flow tests as self- and rapid tests. The rapid expansion of this field requires the design and validation of novel, affordable, and versatile technologies for the easy fabrication of a variety of lateral flow devices. In the present work, we have developed a new, simple, and cost-effective system for the dispensing of reagents on the membranes of lateral flow devices to be used for research purposes. The 3D printing technology is integrated, for the first time, with simple and inexpensive tools such as a technical pen and disposable pipet tips for the construction of the test and the control areas of the devices. We also used this system for the automated fabrication of spots on the membrane for multiplex analysis. The devices were applied for the detection of proteins/antibodies and single- and double-stranded DNA targets. Also, devices with multiple biosensing areas on the membrane were constructed for the simultaneous detection of different analytes. The proposed system is very simple, automated, and inexpensive and has provided rapid and reproducible construction of lateral flow devices. Compared to a commercially available automated dispenser, the devices showed similar detection capabilities and reproducibility in various real samples. Moreover, contrary to the existing dispensers, the proposed system does not require any gas or costly precision pumps and syringes for the deposition. In conclusion, the developed 3D printer-based system could be an extremely useful alternative for research laboratories for the construction of lateral flow devices of various assay configurations.

Preparation of an imprinted monolith for field simultaneously selective separation and enrichment of nitrophenols and lead (II) ion in water samples

Imprinted materials have gained significant attention due to its highly specific recognition performance. However, there is no study that using molecule and ion as mixed templates to prepare hybrid imprinted material for simultaneously selective separation and enrichment of molecules and ion. Here, a new strategy employing organic molecule and heavy metal ion as mixed templates to prepare imprinted material was proposed. 2,4-Dinitrophenol (2,4-DNP) and Pb2+ were selected as model templates to in-situ synthesize adsorbent based on imprinted monolith (AIM) in pipette tip. The obtained AIMs were used as the extraction medium of home-made multiple channels in-tip microextraction device (MCMD) for field simultaneously selective entrapment of nitrophenols (NPs) and Pb2+. The selection of functional monomers was well verified with Density function theory. Under the optimal fabrication conditions, the AIM displayed high specific recognition capability towards 2,4-DNP and Pb2+ with imprinted factors 7.31 and 5.20, respectively, and corresponding adsorption capacities were as high as 27.8 mg/g and 20.3 mg/g, respectively. The practicality of proposed AIM/MCMD was evaluated by simultaneously on-site selective capture of NPs and Pb2+ in various environmental waters prior to quantification with HPLC (for NPs) and atomic absorption spectrometry (for Pb2+). Results well evidence that the proposed strategy is feasible in the preparation of imprinted polymer for simultaneously specific extraction of molecules and metal ion, and the developed AIM/MCMD is a useful field sample preparation method for reliable quantification of NPs and Pb2+ in aqueous samples.

CZE-MS peptide mapping: To desalt or not to desalt?

BackgroundIn “shotgun” approaches involving high-performance liquid chromatography or capillary zone electrophoresis (CZE), matrix removal prior to sample analysis is considered as an indispensable tool. Despite the fact that CZE offers a high tolerance towards salts, most publications reported on the use of desalting. There seems to be no clear consensus on the utilization of desalting in the CZE-MS community, most probably due to the absence of works addressing the comparison of desalted and non-desalted digests. Our aim was to fill this research gap using protein samples of varying complexity in different sample matrices. ResultsFirst, standard protein digests were analyzed to build the knowledge on the effect of sample clean-up by solid-phase extraction (SPE) pipette tips and the possible stacking phenomena induced by different sample matrices. Desalting led to a somewhat altered peptide profile, the procedure affected mostly the hydrophilic peptides (although not to a devastating extent). Nevertheless, desalting samples allowed remarkable stacking efficiency owing to their low-conductivity sample background, enabling a so-called field-amplified sample stacking phenomenon. Non-desalted samples also produced a stacking event, the mechanism of which is based on transient-isotachophoresis due to the presence of high-mobility ions in the digestion buffer itself. Adding either extra ammonium ions or acetonitrile into the non-desalted digests enhanced the stacking efficiency. A complex sample (yeast cell lysate) was also analyzed with the optimal conditions, which yielded similar tendencies. SignificanceBased on these results, we propose that sample clean-up in the bottom-up sample preparation process prior to CZE-MS analysis can be omitted. The preclusion of desalting can even enhance detection sensitivity, separation efficiency or sequence coverage.

Eco-friendly miniaturized kapok-fiber supported liquid-phase extraction coupled with gas chromatography-flame ionization detector for high-throughput edible oil preparation and low-cost determination of synthetic antioxidants

The addition of synthetic phenolic antioxidants can effectively delay or prevent oxidation of edible oils. However, excessive use of these antioxidants can have negative impacts, which emphasizes the importance of accurate analysis methods to determine their presence in edible oils. Given the large number of food samples involved in regulatory purposes, there is a need to develop high-throughput and cost-effective detection methods for routine analysis. However, the existing methods are not suitable, indicating a need for further improvement. This study presents a technique that combines miniaturized-kapok fiber-supported liquid-phase extraction (mini-KF-SLE) with gas chromatography-flame ionization detection (GC-FID) to determine synthetic antioxidants like 3-tert-butyl-4-hydroxyanisole, 3,5-di-tert-butyl-4-hydroxytoluene, and tert-butylhydroquinone in edible oils. The mini-KF-SLE device consists of 20mg of kapok fibers placed in a 1mL pipette tip attached to a pipette. The extraction procedure involves three simple steps: loading the oil sample (150mg), adding the extractant (150µL of ACN), and eluting the extractant by applying air pressure using a pipette. The developed mini-KF-SLE-GC-FID method shows satisfactory quantitation ability (R2>0.99) and detection limits of 0.4–0.5µg/g. Moreover, the relative standard deviations for all analytes were below 9.2%, indicating good precise of the method for routine analysis. The proposed method also exhibits excellent environmental friendliness, as evidenced by three widely recognized metrics. Overall, the mini-KF-SLE-GC-FID method provides a convenient, low-cost, environmentally friendly, and easy to automate alternative for routinely determining synthetic antioxidants in edible oils.

Glowing gels and pipettes aplenty: how do commercial stock image banks portray genetic tests?

News stories and patient-facing material about genetic tests are often illustrated by images, but the content of such images and the messages they propagate are rarely scrutinised. Stock image banks were searched to identify a hundred images relating to genetic tests and analysed using a multimodal critical discourse approach, aiming to identify what the images featured, how they were composed, and what they communicated about genetic testing. We found that images tended to focus on technical aspects of sample processing (for example, pipetting) and drew on older technologies (for example slab gel electrophoresis) when representing data arising from genetic tests. Composition choices like focussing images around pipette tips, or emphasising colour or brightness of electrophoretic bands, represented genetic testing as precise, unambiguous and illuminating. Only 7% of images featured a person having a genetic test, and only one image alluded to communication of genetic results. Current popular visual representations of genetic testing rarely highlight the possibility of uncertain or non-diagnostic outcomes, and may contribute to high public expectations of informativeness and certainty from such tests.

Centrifugal Gel Crushing Tips for Gel-Based Proteome Analysis.

We have developed a centrifugal gel-crushing method using a pipet tip. Polyacrylamide gel slices are extruded from the narrowing cavity of a pipet tip by centrifugation in a few minutes to crush them into pieces of appropriate size. The size of the crushed gel could be controlled by several parameters, including centrifugal force and pipet tip cavity. In shotgun proteomics, gel-based LC/MS/MS, so-called GeLC/MS/MS, involves the essential but tedious processes of prefractionation by SDS-PAGE, followed by dicing the entire gel lane into several parts, fine dicing, and in-gel digestion after the diced gel is manually transferred to a microtube. In this study, we developed an alternative way to crush the prefractionated gel slice into optionally small and irregular-shaped gels by centrifugal extrusion of the sliced gel from the narrow cavity of a pipet tip. As a result, we observed improved recovery and reproducibility of digested proteins compared to the conventional method of manual dicing. We believe that this simple and rapid method of crushing polyacrylamide gels, which allows for parallel operations and automation, is useful for GeLC/MS/MS analysis and applicable to other approaches, including top-down proteomics.

Quality control of psychoactive substances in human breastmilk samples from a human milk bank by disposable pipette extraction-gas chromatography-mass spectrometry.

The lack of data regarding infant exposure to drugs of abuse consumed by lactating mothers has become a major health concern. Investigating psychoactive substances and their metabolites in breastmilk is an analytical approach to estimate the rate at which drugs of abuse are excreted and how much infants are exposed to them. In this study, we have developed and validated a GC-MS method using disposable pipette tips for simultaneously extracting ten analytes from breastmilk samples obtained from a milk bank in the city of Ribeirão Preto, Brazil. All the 67 analyzed samples tested negative for all the analytes. This is the first study that has applied disposable pipette extraction to analyze drugs of abuse in breastmilk samples.