Micropipette Tip Research Articles

Metal-Organic Framework Sub-Nanochannels within the Confined Micropipettes: Precise Construction Makes It a Universal Aptamer-Based Sensing Platform.

It is crucial to precisely construct metal-organic framework (MOF) sub-nanochannels at the tip of micro/nanopipettes for fundamental research and sensing applications. The quality of the MOF modification plays a significant role in influencing subsequent research, particularly in sensing applications. In this work, we present a precise method of constructing MOF sub-nanochannels at the tip of glass micropipettes, which serve as a universal aptamer-based sensing platform for the selective detection of proteins. In situ scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS) mapping, and fluorescence microscopy results demonstrate that the synthesized MOF (UiO-66) nanocrystals fully block the orifice of glass micropipettes (UiO-66-GMs) without forming any nanometer-scale cracks and remain confined within the geometric boundaries of the orifice. The terminal phosphate-modified aptamer readily binds to the surface of UiO-66-GMs through metal (Zr)-phosphate coordination, ultimately forming the aptamer sensor (Apt-UiO-66-GMs). The selective quantification of proteins is achieved via a decrease in current resulting from protein binding to the aptamer. Our results indicate that the precisely constructed Apt-UiO-66-GMs sensor enables highly selective and sensitive detection of SARS-CoV-2 nucleocapsid protein and holds potential for real sample detection. Furthermore, given the sharp tip of the micropipets and the external sensing interface we have constructed, our aptamer-based sensing platform also opens avenues for single-cell analysis and in vivo sensing.

Extraction of pesticides from soil using direct-immersion SPME LC-Tips followed by GC–MS/MS: Evaluation and proof-of-concept

A new method was evaluated and developed for the analysis of pesticides in sandy-loam soil by direct-immersion solid phase microextraction (DI-SPME) followed by gas chromatography tandem-mass spectrometry (GC–MS/MS) determination. Ten pesticides were selected based on a literature survey of the compounds reported to be present in EU soils. The extraction was performed using SPME LC-Tips, a new SPME configuration with the coated fibers attached to a disposable and easy-to-handle micropipette tip, which was immersed into a soil slurry made by the addition of an aqueous solution to the soil sample. Ten experimental parameters were evaluated with a Plackett-Burman design, after which the extraction time and percentage of organic solvent in the aqueous extraction were optimized separately. The two fiber chemistries available (PDMS/DVB and C18) were evaluated in parallel for the entire work. In the final method, slurry samples were made by adding an aqueous solution (6 % methanol v/v) to 2 g of soil. The fiber was conditioned and then inserted, for extraction, into the samples, stirred by a magnetic bar. Afterwards, the analytes were desorbed onto 100 µL of methanol. After the addition of analyte protectants (ethylglycerol, gulonolactone, and sorbitol) the extract was injected into the GC–MS/MS system. Isotopically labelled penconazole was used as internal standard. A calibration was performed by extracting spiked soil with analyte concentrations of 0.1–50 µg/kg. Coefficients of determination of the linear calibration were between 0.94–0.98 for the PDMS/DVB and 0.92–0.99 for the C18. Limits of detection range between 0.01–10 µg/kg for the PDMS/DVB and 0.1–10 µg/kg for the C18. Overall, the C18 analytically outperformed the PDMS/DVB but required a longer extraction time (120 min vs 75 min for the PDMS/DVB). This method allows automation and generates low residual toxic waste, having the potential to be introduced as a greener and simpler alternative to currently used sample preparation methodologies.

Preconcentration of nanoplastics using micro-electromembrane extraction across free liquid membranes

Asymmetric micro-electromembrane extraction (µ-EME) based on a free liquid membrane has been evaluated for the preconcentration of nanoplastics. A conical unit (200 µL micropipette tip) enabled the simple and reproducible formation of the required three-phase extraction system consisting of a donor solution (150 µL sample/standard solution), free liquid membrane (FLM; 10 µL 1-pentanol), and an acceptor solution (5 µL of 5 mM phosphate buffer, pH 10.7). After µ-EME, nanoplastics transferred across the FLM into the acceptor solution were quantified using capillary zone electrophoresis with diode array detection. Enrichment factors >20 and extraction recoveries >70 % were achieved for nanoplastics concentrated at 500 V during 5 min. The limit of detection (LOD, S/N = 3) and limit of quantification (LOQ, S/N = 10) of the method using 200 nm sulphonated polystyrene particles as model nanoplastics were 6.00×10−4% (w/v) and 2.00×10−3% (w/v), respectively. Intraday (n = 6) and interday (n = 6) repeatability%RSD for 5.5 × 10−3% (w/v) nanoplastics were 8.5 % and 7.2 %, respectively. µ-EME enabled an efficient sample matrix clean-up and preconcentration of nanoplastics spiked in tea sample matrices. Nanoplastics preconcentrated through the FLM for black tea resulted in an enrichment factor of 20±3.6 (n = 3), with complete sample matrix removal of UV absorbing compounds.

Direct ESI-MS of Ionic Liquids Using High-Pressure Electrospray From Large-Bore Emitters Made of Micropipette Tips.

Electrospray ionization mass spectrometry of neat undiluted ionic liquid (IL) and the analysis of protein with the doping of IL were performed using high-pressure electrospray. The use of disposable micropipette tips as emitters eased the handling of viscous and easy-to-clog samples and improved the reproducibility of the measurement. A high-pressure operation enabled the stable electrospray of the highly conductive IL from these relatively large bore emitters. The measurement of the current-voltage relationship of 1-ethyl-3-methylimidazolium tetrafluoroborate (Emim BF4) revealed an unusual negative differential resistance that has not been seen in the typical atmospheric or high-pressure electrospray. Mass spectrometric analysis of this IL also showed the characteristic response of various ion species with the emitter voltage. When added to the commonly used protein solution, the mass spectrum also showed protein peaks that correspond to the adduction of fluoroboric acid molecules (HBF4).

Automated Piezo-Assisted Sperm Immobilization

Sperm immobilization is a crucial procedure in clinical cell surgery for infertility treatment. Current immobilization is implemented by tapping the sperm tail with a glass micropipette, but its effectiveness is restricted by sperm orientation and ineffective membrane ablation. Ineffective ablation leads to limited release of oocyte activating factors and lowers fertilization rate; and sperm swim in small angles relative to the micropipette tip cannot be tapped due to the risk of damaging the sperm’s genetic materials contained in the sperm head. This paper reports automated piezo-assisted sperm immobilization with enhanced efficacy of cell membrane ablation and sperm orientation control. The designed piezo drill consists of two orthogonal vibration modules to generate controlled micropipette vibration along axial and lateral axes. Through stiffness modeling, the flexure joints guide the motion of the central beam of each vibration module. To achieve sperm orientation control, whirl flow is induced by both axial and lateral vibration of the micropipette tip. To immobilize sperm, only micropipette’s axial vibration is generated to prevent lateral vibration from damaging sperm head. A visual servoing scheme is developed by decoupling sperm wiggling from positioning error for immobilization. Experimental results showed that sperm orientation control by the piezo drill achieved an error of 1.4 <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$^{\circ}$</tex-math> </inline-formula> and a time cost of 2.5 s. Visual servoing with sperm wiggling decoupling achieved a positioning error of 1.7 <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\mu$</tex-math> </inline-formula> m. Furthermore, the piezo-assisted sperm immobilization technique led to effective membrane ablation. With membrane-impermeable stains, it took 5.6 s for the immobilized sperm to be stained after piezo-assisted immobilization, significantly less than 49.2 s by conventional micropipette tapping. <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">Note to Practitioners</i> —This work tackled the challenge of ineffective membrane ablation and orientation limit in clinical cell surgeries. Conventional manual immobilization suffers from low membrane ablation efficacy, which leads to limited release of oocyte activating factors and lowers fertilization rate. Moreover, sperm swim in small angles relative to the micropipette tip cannot be tapped due to the risk of damaging the sperm’s genetic materials contained in the sperm head. In this paper, we propose automation techniques for effective membrane ablation and orientation control of sperm. A clinically compatible piezo drill is developed to generate controllable micropipette motion along both axial and lateral directions. The whirl flow generated by micropipette vibration is employed to rotate sperm, which greatly increased the number of available sperm for immobilization. A visual servoing controller is developed to keep the sperm at the center of field of view for immobilization by decoupling sperm wiggling from positioning error. The developed methods can be generalized to the manipulation of other types of cells. The piezo drill can be used for effective membrane ablation of oocyte, embryo, yeast cell and so on. The orientation control strategy leveraging piezo-induced whirl flow is applicable to non-contact rotation of a variety of microorganism.

Microscale geometrical modulation of PIEZO1 mediated mechanosensing through cytoskeletal redistribution

The microgeometry of the cellular microenvironment profoundly impacts cellular behaviors, yet the link between it and the ubiquitously expressed mechanosensitive ion channel PIEZO1 remains unclear. Herein, we describe a fluorescent micropipette aspiration assay that allows for simultaneous visualization of intracellular calcium dynamics and cytoskeletal architecture in real-time, under varied micropipette geometries. By integrating elastic shell finite element analysis with fluorescent lifetime imaging microscopy and employing PIEZO1-specific transgenic red blood cells and HEK cell lines, we demonstrate a direct correlation between the microscale geometry of aspiration and PIEZO1-mediated calcium signaling. We reveal that increased micropipette tip angles and physical constrictions lead to a significant reorganization of F-actin, accumulation at the aspirated cell neck, and subsequently amplify the tension stress at the dome of the cell to induce more PIEZO1’s activity. Disruption of the F-actin network or inhibition of its mobility leads to a notable decline in PIEZO1 mediated calcium influx, underscoring its critical role in cellular mechanosensing amidst geometrical constraints.

In-tip-fabric phase sorptive extraction (In-tip-FPSE): A proof of concept study for on-site and in-laboratory sample preparation of antidepressants and benzodiazepines

In forensic toxicology, the accurate identification and quantification of drugs are essential for medico-legal investigations. However, the challenge of drug stability complicates this task. On-site extraction (OSE) offers a solution, eliminating the need for sample transportation and storage, reducing degradation risks, and enhancing time, cost, and data quality. In the present work, we propose an innovative approach: In-tip-fabric phase sorptive extraction (In-tip-FPSE), designed for both on-site and in-laboratory sample preparation of antidepressants (ADs) and benzodiazepines (BZDs) from urine and postmortem blood samples. Initially, a sol–gel Carbowax 20 M (CW20M) coated FPSE membrane was prepared using unbleached cotton as the fabric substrate. For the fabrication of the In-tip-FPSE device, two cleaned FPSE membranes (1 cm × 1.5 cm) were positioned inside a 1 mL micropipette tip and fitted into a standard micropipette. The extraction procedure involves repeated aspirating/dispensing cycles (40 times) of the diluted samples within the micropipette tip to facilitate efficient extraction onto the FPSE membrane. Subsequently, elution is achieved by aspirating 1 mL of methanol (MeOH), followed by vertical positioning of the micropipette for 10 min, after which the eluted MeOH is dispensed into a GC–MS vial for analysis. Optimization of the In-tip-FPSE procedure involved exploring parameters such as the type and size of membrane, number of aspirating/dispensing cycles, type and volume of elution solvent, sample pH, and ionic strength. Under optimized conditions, the limit of quantification (LOQ) ranged from 33-41 µg/L and 42–49 µg/L for urine and blood, respectively. The relative standard deviation for intra and inter-day precision was less than 4 % and 10 %, respectively. The method exhibited excellent linearity in the range of 5–5000 µg/L for both matrices with a coefficient of determination (R2) in the range of 0.998 – 0.999. Absolute recovery (AR%) values for this method in real samples ranged from 85%-110 %. Furthermore, the practicality, greenness, and sustainability of the In-tip-FPSE method were assessed, demonstrating its superiority over previously reported in-laboratory methods. Finally, the method was successfully applied for OSE of target analytes from urine samples and in-laboratory analysis of postmortem blood samples.

Ion-Selective Micropipette Sensor for In Vivo Monitoring of Sodium Ion with Crown Ether-Encapsulated Metal-Organic Framework Subnanopores.

In vivo sensing of the dynamics of ions with high selectivity is essential for gaining molecular insights into numerous physiological and pathological processes. In this work, we report an ion-selective micropipette sensor (ISMS) through the integration of functional crown ether-encapsulated metal-organic frameworks (MOFs) synthesized in situ within the micropipette tip. The ISMS features distinctive sodium ion (Na+) conduction and high selectivity toward Na+ sensing. The selectivity is attributed to the synergistic effects of subnanoconfined space and the specific coordination of 18-crown-6 toward potassium ions (K+), which largely increase the steric hindrance and transport resistance for K+ to pass through the ISMS. Furthermore, the ISMS exhibits high stability and sensitivity, facilitating real-time monitoring of Na+ dynamics in the living rat brain during spreading of the depression events process. In light of the diversity of crown ethers and MOFs, we believe this study paves the way for a nanofluidic platform for in vivo sensing and neuromorphic electrochemical sensing.

Charge transfer across the ionic liquid/oil interface: Facilitated ion transfer and electron transfer

Facilitated ion transfer (FIT) and electron transfer (ET) across the ionic liquid (IL)/oil (O) interface between ethylamine nitrate (EAN) and 1,4-dichlorobutane (DCB) have been electrochemically studied. Cyclic voltammograms (CVs) for the FIT of Li+ cation in IL by dicyclohexano-18-crown-6 (DCH18C6) across the IL/O interface formed at a micropipette tip show that the onset of the FIT current is significantly shifted due to the facilitation beyond the potential window limit and that the current is proportional to the concentration of DCH18C6 in O limited by the diffusion of DCH18C6 inside the micropipette. CVs for the ET from supporting electrolyte anions in O to NO3−, the IL anion of EAN, across the IL/O interface studied using a closed-bipolar cell show that NO3− in EAN has an ability to oxidize tetraphenylborate ions in O but not tetrakis[3,5-bis(trifluoromethyl)phenyl]borate ions, the latter of which have more oxidation resistance.

Development of Vibratory Microinjection System for Instantaneous Cell Membrane Piercing in Cytoplasmic Microinjection into Fertilized Eggs

To complete microinjection as quickly as possible, we have developed Vibratory Microinjection Systems (VMSs) that vibrate a micropipette in its longitudinal direction and can significantly reduce the time needed for pronuclear microinjection compared to ordinary (non-vibratory) microinjection. The longest breakdown of the time is the time required to pierce the cell membrane and the pronuclear membrane simultaneously. Because cytoplasmic microinjection, which pierces the cell membrane alone, is far more difficult and time-consuming than pronuclear microinjection, we next aimed to develop a VMS capable of penetrating the cell membrane instantly. In this new and latest version, two types of ultrasonic-wave vibrators were developed: the first for commercially available micropipettes (Femtotip) and the second for self-made micropipettes. The two vibrators differ only in their airtight structure, where the micropipettes connect to their respective vibrators: a female screw plus O-ring for the first vibrator (VMS6_1) and a silicone–-rubber tube for the second (VMS6_2). The tube-type joint used in VMS6_2 only slightly damped or amplified vibrations from the vibrator to the micropipette tip, propagating them much more accurately than the screw-type joint in VMS6_1. In addition, VMS6_2 significantly shortened the time taken to pierce the cell membrane of a fertilized egg: an average of 1.52 s (N = 410) vs. 3.62 s (N = 65) in VMS6_1. The VMS6_2 group achieved a piercing time of zero in 86.1% of the allocated eggs, while only 10.8% of the VMS6_1 group did. In each vibrator, we also compared vibratory microinjection (VM; N = 475) and ordinary microinjection (OM; N = 457), which uses injection pressure in place of vibration. None of the eggs in the OM group achieved the zero-second piercing time. Compared to the OM, the VM group showed a significantly shorter piercing time, 1.80 vs. 10.69 s on average, and a significantly better survival rate, 90.3 vs. 81.8% on average. VMS6_2 not only improved on the already demonstrated superiority of VM to OM but also enabled instantaneous piercing of the cell membrane.

TidyTron: Reducing lab waste using validated wash-and-reuse protocols for common plasticware in Opentrons OT-2 lab robots

Every year biotechnology labs generate a combined total of ∼5.5 million tons of plastic waste. As the global bioeconomy expands, biofoundries will inevitably increase plastic consumption in-step with synthetic biology scaling. Decontamination and reuse of single-use plastics could increase sustainability and reduce recurring costs of biological research. However, throughput and variable cleaning quality make manual decontamination impractical in most instances. Automating single-use plastic cleaning with liquid handling robots makes decontamination more practical by offering higher throughput and consistent cleaning quality. However, open-source, validated protocols using low-cost lab robotics for effective decontamination of plasticware—facilitating safe reuse—have not yet been developed. Here we introduce and validate TidyTron: a library of protocols for cleaning micropipette tips and microtiter plates that are contaminated with DNA, E. coli, and S. cerevisiae. We tested a variety of cleaning solutions, contact times, and agitation methods with the aim of minimizing time and cost, while maximizing cleaning stringency and sustainability. We tested and validated these cleaning procedures by comparing fresh (first-time usage) versus cleaned tips and plates for contamination with cells, DNA, or cleaning solutions. We assessed contamination by measuring colony forming units by plating, PCR efficiency and DNA concentration by qPCR, and event counts and debris by flow cytometry. Open source cleaning protocols are available at https://github.com/PlantSynBioLab/TidyTron and hosted on a graphical user interface at https://jbryantvt.github.io/TidyTron/.

In Situ Probing the Short-Lived Intermediates in Visible-Light Heterogeneous Photocatalysis by Mass Spectrometry.

Visible-light-mediated heterogeneous photocatalysis has recently emerged as an environmentally friendly and energy-sustainable alternative for organic transformations. Despite the advancements in developing wide varieties of photocatalysts during the past decades, the accurate probing and identification of the photogenerated species, especially the short-lived radical intermediates, are still challenging. In this work, we reported a hybrid ion emitter that integrated with a pico-liter heterogeneous photocatalytic reactor, which was fabricated by depositing the photocatalyst (e.g., TiO2) into the front tip of a quartz micropipette. Benefited from the dual-function feature of the hybrid micropipette (i.e., a clog-free tip-confined pico-liter reactor for heterogeneous photocatalysis and an ion emitter for nanoelectrospray ionization), sensitized photoredox reactions at the catalyst-solution interface can be triggered upon visible-light irradiation using a cheap LED laser (453 nm), and the newly produced transient radical intermediates can be rapidly transformed into gaseous ions for mass spectrometric identification. Using this novel low-delay coupling device, photogenerated intermediates, including the cationic radicals produced during the photooxidation of anilines and the anionic radicals produced during the photoreduction of quinones, were successfully captured by mass spectrometry. We believe that our hybrid photochemical microreactor/ion emitter has provided a new and powerful tool for exploring the complicated heterogeneous photochemical processes, especially their ultrafast initial transformations.

Characteristics of high-pressure nanoESI-MS using standard and chromatographically packed (ZipTip) micropipette tips as emitters for highly conductive solutions

Voltage-driven electrospray of highly conductive solutions under high pressure without gaseous breakdown enabled a low flow rate nanoESI using an emitter of large inner diameters such as a commercial micropipette tip. This feature was previously exploited to observe unique ionization responses at flow rates close to their minimum values (Chem. Sci.14, 4506–4515 &Anal. Chem.94, 16,015–16022). Here, we studied the properties of such pipet tip sprayers at a higher spray current up to >2 μA with flow rates approaching their maximum values. Normal pipet tips with 0.37 mm i.d. and tips packed with chromatographic particles (ZipTip, i.d. 0.44 mm) were used. The nanoflow rate was tuned using emitter voltage with spray current as the monitoring signal for flow rate. The spray current I and flow rate Q relationship followed the I∝Q1/2 law for I < 1 μA. Above that point, the Taylor cone was still stable but the spray current increased at a higher rate with the flow rate. The packed pipet tips have a larger flow resistivity, required a higher onset voltage, and interestingly, supported a higher stable spray current and flow rate. HP-nanoESI-MS of protein and oligosaccharide showed variation in charge state and ion intensity with the spray current. The direct nanoESI of extraction solution from the ZipTip right after the desalting and concentrating procedures were also demonstrated.

A novel device for swift and efficient CD44 protein digestion of pipette tips in human serum

For molecular diagnostics in modern biomedical research, electrospray ionisation mass spectrometry (ESI-MS) based on proteome profiling is important. Now a days, sample preparation such as proteolysis and protein extraction remain incredibly challenging and inefficient. Recent sample-preparation methods based on micro tips show promising results toward the aim “a proteome in an hour”. Proteolysis at the tip, is still infrequently observed and does not represent the processing of complex bio-samples. In this study, we outline a unique technique for detecting and extracting human serum CD44 biomarkers by ligand–protein interactions. This method employs macropores silica particles (MPSP) or (MOSF) modified with hyaluronic acid (HA). In order to assist in the profile of the human serum proteome, we limitations of immunoassays for rapid and multimodal proteolysis. For effective in situ proteolysis, in micropipette tips, MPSP were designed as nanoreactors with variable pore size and surface chemistry. In MS-based bottom-up proteome analysis, the device as-built demonstrated favourable sensitivity (LOD of 0.304 ± 0.007 ng/mL and LOQ of 0.973 ± 0.054 ng/mL), selectivity, durability (at −20 °C for 2 months), reuse (at least 10 times), and minimal memory impact. In addition, we examined into specific surface chemistries of nanoparticles for the absorption of proteins in serum and profiled the HA-binding serum proteome, setting a new preliminary benchmark for future databases. Our study not only helped establish a new platform for extracting/detection of CD44 and identifying the HA-binding proteome, but it also offered design recommendations for ligand affinity-based techniques for the antibody-free study of serum biomarkers with a view towards diagnostic applications.

A Cartilaginous Construct with Bone Collar Exerts Bone-Regenerative Property Via Rapid Endochondral Ossification.

Three-dimensional clumps of mesenchymal stem cells (MSCs)/extracellular matrix (ECM) complexes (C-MSCs) can be implanted into tissue defects with no artificial scaffolds. In addition, the cellular properties and characteristics of the ECM in C-MSCs can be regulated in vitro. Most bone formation in the developmental and healing process is due to endochondral ossification, which occurs after bone collar formation surrounding cartilage derived from MSCs. Thus, to develop a rapid and reliable bone-regenerative cell therapy, the present study aimed to generate cartilaginous tissue covered with a mineralized bone collar-like structure from human C-MSCs by combining chondrogenic and osteogenic induction. Human bone marrow-derived MSCs were cultured in xeno-free/serum-free (XF) growth medium. Confluent cells that formed cellular sheets were detached from the culture plate using a micropipette tip. The floating cellular sheet contracted to round clumps of cells (C-MSCs). C-MSCs were maintained in XF-chondro-inductive medium (CIM) and XF-osteo-inductive medium (OIM). The biological and bone-regenerative properties of the generated cellular constructs were assessed in vitro and in vivo. C-MSCs cultured in CIM/OIM formed cartilaginous tissue covered with a mineralized matrix layer, whereas CIM treatment alone induced cartilage with no mineralization. Transplantation of the cartilaginous tissue covered with a mineralized matrix induced more rapid bone reconstruction via endochondral ossification in the severe combined immunodeficiency mouse calvarial defect model than that of cartilage generated using only CIM. These results highlight the potential of C-MSC culture in combination with CIM/OIM to generate cartilage covered with a bone collar-like structure, which can be applied for novel bone-regenerative cell therapy.

Micropipette tip intubation in rats as a replacement for conventional endotracheal tube intubation.

Endotracheal intubation is often necessary in the course of animal experiments, especially in craniofacial surgery. However, endotracheal intubation can be a major burden in this context. The authors performed simple and cost-saving method using a 200 μL yellow micropipette tip, and the success of this method was demonstrated by X-ray and autopsy. We used a total of 30 rats. After the rats were fixed with a plaster, the trachea and vocal cords were visualized with the tongue pulled back. Under direct visualization of the vocal cords, a curving micropipette tip was advanced into the trachea. This method can be learned quickly and applied successfully by general experimenters. We successfully intubated all 30 rats without any complications. The success rate of micropipette tip intubation was 100%. This procedure was performed by one experimenter within 2 to 3 minutes after induction of anesthesia. We demonstrated its superiority by X-ray and autopsy. Herein, we describe endotracheal intubation of rats using micropipette tips. To the best of our knowledge, this method is novel and represents the simplest and most efficient means of intubation in rats, providing an alternative to conventional endotracheal intubation.

On-Demand Portable Paper-Based Electrospray Ionization Mass Spectrometry for High-Sensitivity Analysis of Complex Samples.

Paper spray ionization has been demonstrated to be the most promising substrate-based source, but this technique suffers from the low desorption efficiency of target compounds and poor portability. In the current study, we describe a portable paper-based electrospray ionization (PPESI) in which a piece of triangle paper and adsorbent are packed sequentially into a modified disposable micropipette tip. This source not only captures the feature of paper spray and adsorbent for highly efficient suppression of sample matrixes for target compound analysis but also takes advantage of a micropipette tip to prevent spray solvent from rapid evaporation. The performance of developed PPESI depends on the type and amount of packed adsorbent, paper substrate, and spray solvent and applied voltage. Moreover, by contrast to other related sources, the analytical sensitivity and the spray duration of PPESI in tandem with MS have been improved by factors of 2.8-32.3 and 2.0-13.3, respectively. Based on its high accuracy (>96%) and precision (less than 3% relative standard deviation), the PPESI coupled to a mass spectrometer has been used to determine diverse therapeutic drugs and pesticides in complex biological (e.g., whole blood, serum, and urine) and food (e.g., milk and orange juice) matrixes, and the limits of detection and quantification were 2-4 pg mL-1 and 7-13 pg mL-1, respectively. Taking the portability, high sensitivity, and repeatability, the technique may be a promising alternative for complex sample analysis.

Sensor-on-Microtips: Design and Development of Hydrothermally Grown ZnO on Micropipette Tips as a Modified Working Electrode for Detection of Glucose.

Miniaturization of electrochemical components has become less common in the last decade, with the focus predominantly being the design and development of state-of-the-art microelectrodes for achieving small volume analysis of samples. However, such microelectrodes involve cumbersome processing procedures to convert the base material for the required application. A potential paradigm shift in such miniaturization could be achieved by using cheaper alternatives such as plastics to build electrochemical components, such as micropipette tips made of polypropylene, which are commercially available at ease. Hence, this work presents the design of an electrochemical working electrode based upon a micropipette tip, involving minimal processing procedures. Furthermore, such a working electrode was realized by sputtering silver onto a bare micropipette tip using a radio-frequency sputtering technique, to obtain electrical contacts on the tip, followed by hydrothermal growth of ZnO, which acted as the active electrode material. The ZnO nanostructures grown on the micropipette tip were characterized for their morphology and surface properties using a scanning electron microscope (SEM), laser microscope, Raman spectrometer, and X-ray photoelectron spectrometer (XPS). The developed micropipette tip-based electrode was then used as the working electrode in a three-electrode system, wherein its electrochemical stability and properties were analyzed using cyclic voltammetry (CV). Furthermore, the above system was used to detect glucose concentrations of 10-200 µM, to evaluate its sensing properties using amperometry. The developed working electrode exhibited a sensitivity of 69.02 µA/µM cm-2 and limit of detection of 67.5 µM, indicating the potential for using such modified micropipette tips as low-cost miniaturized sensors to detect various bio-analytes in sample solutions.

Functionalization of micropipette tips with hydrophobin-laccase chimera and application to the electrochemical determination of caffeic acid in tea samples

Performing on-site measurements is one of the main challenges of current (bio)chemical analysis. Micropipette tips, which are among the most common utilities in laboratories, can be useful and cheap elements for designing out-of-the-box innovative analytical biodevices. Furthermore, most of the analytical properties of these bioplatforms depend on the immobilization of the recognition element (e.g., enzymes). In this context, we have recently designed a chimeric protein in which laccase PoxA1b from the fungus Pleurotus ostreatus has been genetically fused to the class I hydrophobin Vmh2. The ability to self-assemble at hydrophobic-hydrophilic interfaces of hydrophobin, allows an easy laccase immobilization. In this work, the Vmh2-PoxA1b fusion protein was immobilized into common polypropylene micropipette tips to develop an innovative enzymatic bioplatform that can produce fast results in a simple way. As proof of applicability, the platform was applied to the determination of caffeic acid by enzymatic oxidation and further electrochemical reduction of the product on a screen-printed electrode, achieving a limit of detection of 1.4·10−6 M. It was then used for determining caffeic acid in tea samples.

COVID-19 Detection Using a 3D-Printed Micropipette Tip and a Smartphone.

The COVID-19 pandemic has caused over 7 million deaths worldwide and over 1 million deaths in the US as of October 15, 2022. Virus testing lags behind the level or availability necessary for pandemic events like COVID-19, especially in resource-limited settings. Here, we report a low cost, mix-and-read COVID-19 assay using a synthetic SARS-CoV-2 sensor, imaged and processed using a smartphone. The assay was optimized for saliva and employs 3D-printed micropipette tips with a layer of monoclonal anti-SARS-CoV-2 inside the tip. A polymeric sensor for SARS-CoV-2 spike (S) protein (COVRs) synthesized as a thin film on silica nanoparticles provides 3,3',5-5'-tetramethylbenzidine responsive color detection using streptavidin-poly-horseradish peroxidase (ST-poly-HRP) with 400 HRP labels per molecule. COVRs were engineered with an NHS-PEG4-biotin coating to reduce nonspecific binding and provide affinity for ST-poly-HRP labels. COVRs binds to S-proteins with binding strengths and capacities much larger than salivary proteins in 10% artificial saliva-0.01%-Triton X-100 (as virus deactivator). A limit of detection (LOD) of 200 TCID50/mL (TCID50 = tissue culture infectious dose 50%) in artificial saliva was obtained using the Color Grab smartphone app and verified using ImageJ. Viral load values obtained in 10% pooled human saliva spiked with inactivated SARS-COV-2 virus gave excellent correlation with viral loads obtained from qPCR (p = 0.0003, r = 0.99).