Advantages of stereolithographic 3D printing in the fabrication of the Affiblot device for dot-blot assays.

In stereolithographic (SLA) 3D printing, objects are constructed by exposing layers of photocurable resin to UV light. It is a highly user-friendly fabrication method that opens a possibility for technology sharing through CAD file online libraries. Here, we present a prototyping procedure of a microfluidics-enhanced dot-blot device (Affiblot) designed for simple and inexpensive screening of affinity molecule characteristics (antibodies, oligonucleotides, cell receptors, etc.). The incorporation of microfluidic features makes sample processing user-friendly, less time-consuming, and less laborious, all performed completely on-device, distinguishing it from other dot-blot devices. Initially, the Affiblot device was fabricated using CNC machining, which required significant investment in manual post-processing and resulted in low reproducibility. Utilization of SLA 3D printing reduced the amount of manual post-processing, which significantly streamlined the prototyping process. Moreover, it enabled the fabrication of previously impossible features, including internal fluidic channels. While 3D printing of sub-millimeter microchannels usually requires custom-built printers, we were able to fabricate microfluidic features on a readily available commercial printer. Open microchannels in the size range 200-300 µm could be fabricated with reliable repeatability and sealed with a replaceable foil. Economic aspects of device fabrication are also discussed.

Preparative agarose gel electrophoresis for reducing matrix interferences of organoid cell medium prior to LC-MS analysis of insulin.

Organoids are 3D cell cultures with microanatomies mimicking aspects of real organs, useful for e.g. animal-free studies of development, disease, and drug discovery. The cell medium of organoid models of Langerhans islets, regulating blood glucose levels by insulin secretion, can be analyzed by liquid chromatography-mass spectrometry (LC-MS). However, organoid medium complexity is a major challenge, as matrix interferences can reduce sensitivity and selectivity, even with optimized LC-MS conditions. By applying preparative agarose gel electrophoresis-electrodialysis (PGE-ED), we were able to decrease the cell medium background signal, allowing for reduced interferences affecting LC-MS analysis of human insulin.

A new device for online nanoscale sampling and capillary electrophoresis analysis of plant sap composition.

In this work, an online sampling of plant xylem sap combined with an efficient (CE)-based method was developed and applied to study the kinetics of changes in the sap composition and to assess plant fitness under stress conditions comprehensively. A laboratory-built CE device was developed to provide online sampling and CE analysis of various ionogenic species in the sap during plant stress response. The rapid online sampling and short CE analysis time allow for real-time monitoring of changes in sap constituents in the living plant during the stress response. The developed device was successfully used to analyze chloride, nitrate, and sulfate ions in the plant xylem during the salt stress or stress caused by nitrate deficiency within short time scales.

Nanospray-assisted deposition of silver nanoparticles for mapping of a peptide in nanofibrous layers via surface-enhanced Raman spectrometry.

Surface-enhanced Raman spectrometry (SERS) is a universal detection tool identifying molecules via vibrations of their chemical bonds. Its function requires the close localization of metal nanostructures and the analyte. In this work, we present a lab-made instrumentation for the deposition of silver nanoparticles on a strongly hydrophilic nanofibrous composite via a nanospray for SERS mapping of an incorporated peptide. The nanospray-sample distance was revealed as the most crucial parameter since it directly influences the moisture of the deposited colloid. Residual water was recognized as a sensitivity enhancer. Additionally, we continuously introduced a solution of sodium chloride to the colloid increasing its ionic strength, which formed a more homogeneous profile of the deposit. After the deposition process, the treated sample was scanned via a SERS laser and the collected Raman spectra were transformed into a distribution map of the peptide at a concentration of 5 µg/g.

Microfluidic device integrating single-cell extraction and electrical lysis for mass spectrometry detection of intracellular compounds.

Analysis of cellular composition and metabolism at a single-cell resolution allows gaining more information about complex relationships of cells within tissues or whole living organisms by resolving the variance stemming from the cellular heterogeneity. Mass spectrometry (MS) is a perfect analytical tool satisfying the demanding requirements of detecting and identifying compounds present in such ultralow-volume samples of high chemical complexity. However, the method of sampling and sample ionization is crucial in obtaining relevant information. In this work, we present a microfluidic sampling platform that integrates single-cell extraction from MS-incompatible media with electrical cell lysis and nanoESI-MS analysis of human erythrocytes. Hemoglobin alpha and beta chains (300 amol/cell) were successfully identified in mass spectra of single-erythrocyte lysates.

Preparative protein concentration from biofluids by epitachophoresis.

With increasing demands on protein analyses in complex biological matrices, the insistence on developing new sample preparation techniques is rising. Recently, we introduced a new displacement electrophoresis technique (epitachophoresis) and instrumentation for preparative concentration and cleaning of DNA samples. This work describes the possibility of applying this device to protein samples. We have developed a method for the epitachophoretic concentration of proteins in a cationic mode and tested it by concentrating and collecting the protein zones from complex biological matrices (urine and growth medium). Under optimized conditions, we have obtained recoveries up to 99%. Furthermore, the applicability of the developed method was proven by concentrating and collecting the cytochrome c zone from a HeLa cell line growth medium, where the protein cytochrome c was released during cell apoptosis.

Absolute Counting Method with Multiplexing Capability for Estimating the Number Concentration of Nanoparticles Using Anisotropically Collapsed Gels.

Number concentration─the number of nanoparticles in a given volume─is an important characteristic of any nanoparticle dispersion. However, its estimation for small nanoparticles (∼30 nm) is generally challenging. We introduce an absolute and widely applicable method for analyzing aqueous dispersions of nanoparticles. An innovative immobilization of nanomaterials in the anisotropically collapsed agarose gel is pioneered, followed by optical microscopy and nanoparticle counting. The number of counted nanoparticles is inherently coupled with sampled volume (517 pL) and translates to the number concentration. Photon-upconversion, fluorescence, bright-field, and dark-field microscopy techniques have been proven applicable and used for imaging lanthanide-doped photon-upconversion nanoparticles, their bioconjugates with antibodies, silica dye-doped fluorescent nanoparticles, quantum dots, and pure silica submicron particles. The precision and linearity were characterized by constructing a dilution series of photon-upconversion nanoparticles. The limit of detection was 2.0 × 106 mL-1, and the working range was from 4.4 × 107 to 2.2 × 1010 mL-1. The quantification of nanoparticle clusters was achieved by a thorough analysis of the micrographs. The accuracy was confirmed using gravimetric analysis and transmission electron microscopy as a reference. Multiplexed detection of two nanoparticle types in a mixed dispersion was feasibly demonstrated. The low thickness of the collapsed gel (<1 µm) supported extremely sensitive imaging. This was proven by imaging Tm3+-doped photon-upconversion nanoparticles (17 nm hydrodynamic diameter) with a nanoparticle emission rate of only ∼900 photons/s at a wavelength of 800 nm (excitation wavelength 976 nm).

Microfabricated Liquid Junction Capillary Electrophoresis-Mass Spectrometry Interface.

Coupling of capillary electrophoresis (CE) with mass spectrometry (MS) represents a powerful combination for performing rapid, efficient, and sensitive analysis of a variety of compounds. Here we describe a construction, operation, and application of a microfabricated liquid junction CE-MS interface. The interface is designed as a microfabricated unit with an integrated liquid junction and electrospray tip made from polyimide, which is positioned in a plastic connection block securing the separation CE capillary and attachable to the CE instrument. The application was demonstrated by CE-MS analysis of dextran oligomers labeled by (2-aminoethyl)trimethylammonium (AETMA) salt.

Bioconjugates of photon-upconversion nanoparticles for cancer biomarker detection and imaging.

The detection of cancer biomarkers in histological samples and blood is of paramount importance for clinical diagnosis. Current methods are limited in terms of sensitivity, hindering early detection of disease. We have overcome the shortcomings of currently available staining and fluorescence labeling methods by taking an integrative approach to establish photon-upconversion nanoparticles (UCNP) as a powerful platform for cancer detection. These nanoparticles are readily synthesized in different sizes to yield efficient and tunable short-wavelength light emission under near-infrared excitation, which eliminates optical background interference of the specimen. Here we present a protocol for the synthesis of UCNPs by high-temperature co-precipitation or seed-mediated growth by thermal decomposition, surface modification by silica or poly(ethylene glycol) that renders the particles resistant to nonspecific binding, and the conjugation of streptavidin or antibodies for biological detection. To detect blood-based biomarkers, we present an upconversion-linked immunosorbent assay for the analog and digital detection of the cancer marker prostate-specific antigen. When applied to immunocytochemistry analysis, UCNPs enable the detection of the breast cancer marker human epidermal growth factor receptor 2 with a signal-to-background ratio 50-fold higher than conventional fluorescent labels. UCNP synthesis takes 4.5 d, the preparation of the antibody-silica-UCNP conjugate takes 3 d, the streptavidin-poly(ethylene glycol)-UCNP conjugate takes 2-3 weeks, upconversion-linked immunosorbent assay takes 2-4 d and immunocytochemistry takes 8-10 h. The procedures can be performed after standard laboratory training in nanomaterials research.

A novel temperature-controlled open source microcontroller based sampler for collection of exhaled breath condensate in point-of-care diagnostics.

Exhaled breath condensate (EBC) is an attractive, non-invasive sample for clinical diagnostics. During EBC collection, its composition is influenced by the collection temperature, a factor that is often not thoroughly monitored and controlled. In this study, we assembled a novel, simple, portable, and inexpensive device for EBC collection, able to maintain a stable temperature at any value between -7 °C and +12 °C. The temperature was controlled using a microcontroller and a thermoelectric cooler that was employed to cool the aluminum block holding the glass tube or the polypropylene syringe. The performance of the novel sampler was compared with the passively cooled RTube™ and a simple EBC sampler, in which the temperature was steadily increasing during sampling. The developed sampler was able to maintain a stable temperature within ±1 °C. To investigate the influence of different sampling temperatures (i.e., +12, -7, -80 °C) on the analyte content in EBC, inorganic ions and organic acids were analyzed by capillary electrophoresis with a capacitively coupled contactless conductivity detector. It was shown that the concentration of metabolites decreased significantly with decreasing temperature. The portability and the ability to keep a stable temperature during EBC sampling makes the developed sampler suitable for point-of-care diagnostics.

Epitachophoresis is a novel versatile total nucleic acid extraction method.

Epitachophoresis is a novel next generation extraction system capable of isolating DNA and RNA simultaneously from clinically relevant samples. Here we build on the versatility of Epitachophoresis by extracting diverse nucleic acids ranging in lengths (20 nt-290 Kbp). The quality of extracted miRNA, mRNA and gDNA was assessed by downstream Next-Generation Sequencing.

Affiblot: a dot blot-based screening device for selection of reliable antibodies.

The key factor in the development of antibody-based assays is to find an antibody that has an appropriate affinity, high specificity, and low cross-reactivity. However, this task is not easy to carry out since the research antibodies on the market may suffer from low specificity and reproducibility. Here, we report on a palm-sized dot blot-based device, called the affiblot, that has a specially designed lid that allows simultaneous semi-quantitative comparison of up to five antibodies from different suppliers regarding their affinity/avidity, cross-reactivity, and batch-to-batch reliability. The only required peripheral equipment is a vacuum pump, a camera, and densitometry software. The affiblot device was tested for its functionality and its measurements were compared against those obtained by standard dot blot and ELISA. The benefit over these methods, when various antibodies are evaluated, is in its simplicity. It allows easy antigen deposition, fast application and the discarding of the solutions, a compact undivided membrane, and therefore significant decrease of labor. The device was tested with specific anti-ApoE, anti-EpCAM, anti-Salmonella, anti-E. coli, and anti-Listeria antibodies from different suppliers. Their properties were compared for their ability to interact specifically with antigen and/or non-target structures and the best-suited antibody for the intended application was identified.

3D printed device for epitachophoresis.

Polyacrylamide or agarose gels are the most frequently used sieving and stabilizing media in slab gel electrophoresis. Recently, we have introduced a new electrophoretic technique for concentration/separation of milliliter sample volumes. In this technique, the gel is used primarily as an anticonvection media eliminating liquid flow during the electromigration. While serving well for the liquid stabilization, the gels can undergo deformation when exposed to a discontinuous electrolyte buffer system used in epitachophoresis. In this work, we have explored 3D printing to form rigid stabilizing manifolds to minimize liquid flow during the epitachophoresis run. The whole device was printed using the stereolithography technique from a low water-absorbing resin. The stabilizing manifold, serving as the gel substitute, was printed as a replaceable composite structure preventing electrolyte mixing during the separation. Different geometries of the 3D printed stabilizing manifolds were tested for use in concentrating ionic sample components without spatial separation. The presented device can focus analytes from 3 or 4 mL of the sample to 150 µL or less, depending on the collection cup size. With the 150 µL collection cup, this represents the enrichment factor from 20 to 27. The time of concentration was from 15 to 25 min, depending on stabilization media and power used.

High-resolution Arduino-based data acquisition devices for microscale separation systems.

In this work, we have designed, constructed, and evaluated simple, inexpensive open-source data acquisition systems based on various analog-to-digital converter modules (ADS 1115, MCP 3424, LTC 2400, with resolution from 16 to 24-bit) and a miniature Arduino Nano ™ microcontroller. The constructed data acquisition systems provide excellent performance and are comparable to a commercial, 24-bit device. We provide full schematics and corresponding source codes so that analytical chemists can easily construct any of the developed systems without extensive electronic or programming knowledge. The 24-bit LTC 2400 based device provided the best and comparable performance to a commercial, high-end 24-bit sigma to delta converter (ORCA 2800) at a fraction of cost (less than 50 USD compared to 870 USD for the commercial counterpart). The excellent performance was verified using a capillary electrophoresis system with contactless conductivity detection and separation of inorganic ions in clinical skin wipe and tap water samples.

Application of capillary electrophoresis-nano-electrospray ionization-mass spectrometry for the determination of N-nitrosodimethylamine in pharmaceuticals.

After a presence of highly hepatotoxic and potentially carcinogenic N-nitrosodimethylamine was detected in certain lots of sartan, ranitidine, metformin, and other pharmaceuticals, local regulatory authorities issued recalls of suspected products, and concerns of the pharmacotherapy safety were widely discussed. Since then, testing of a representative sample of each produced lot of these pharmaceuticals is required as a part of quality control processes. Hence, an interface-free CE-nanoESI system coupled with MS detection was employed for the development of a simple and economical method for quantitative detection of this contaminant in the valsartan drug substances and finished formulations used as model matrices. In this arrangement, a fused-silica capillary was used as both a separation column and a nanoESI emitter providing high ionization efficiency and sensitivity. The optimized procedure was found to have sufficient selectivity, linearity, accuracy, and precision. The established LOD and LOQ values were 0.3 and 1.0 ng/mL, respectively. The practical applicability of the method was tested by analyses of commercially available Valsacor® tablets. The results obtained prove that the developed procedure represents a promising alternative to currently available GC- and LC-based methods. Furthermore, after an adjustment of the separation conditions, the CE-nanoESI/MS system can be conceptually used for the determination of NDMA in other suspected pharmaceuticals.

Electrospray: More than just an ionization source.

Electrospraying (ES) is a potential-driven process of liquid atomization, which is employed in the field of analytical chemistry, particularly as an ionization technique for mass spectrometric analyses of biomolecules. In this review, we demonstrate the extraordinary versatility of the electrospray by overviewing the specifics and advanced applications of ES-based processing of low molecular mass compounds, biomolecules, polymers, nanoparticles, and cells. Thus, under suitable experimental conditions, ES can be used as a powerful tool for highly controlled deposition of homogeneous films or various patterns, which may sometimes even be organized into 3D structures. We also emphasize its capacity to produce composite materials including encapsulation systems and polymeric fibers. Further, we present several other, less common ES-based applications. This review provides an insight into the remarkable potential of ES, which can be very useful in the designing of innovative and unique strategies.

Capillary electrophoretic methods for quality control analyses of pharmaceuticals: A review.

Capillary electrophoresis represents a promising technique in the field of pharmaceutical analysis. The presented review provides a summary of capillary electrophoretic methods suitable for routine quality control analyses of small molecule drugs published since 2015. In total, more than 80 discussed methods are sorted into three main sections according to the applied electroseparation modes (capillary zone electrophoresis, electrokinetic chromatography, and micellar, microemulsion, and liposome-electrokinetic chromatography) and further subsections according to the applied detection techniques (UV, capacitively coupled contactless conductivity detection, and mass spectrometry). Key parameters of the procedures are summarized in four concise tables. The presented applications cover analyses of active pharmaceutical ingredients and their related substances such as degradation products or enantiomeric impurities. The contribution of reported results to the current knowledge of separation science and general aspects of the practical applications of capillary electrophoretic methods are also discussed.

Photon-upconversion barcode for monitoring an enzymatic reaction with a fluorescence reporter in droplet microfluidics.

We report luminescent photon-upconversion barcodes for indexing the chemical content of droplets. The barcode is compatible with the simultaneous detection of fluorescence. The encoding and decoding of the initial concentration of enzyme ß-galactosidase and substrate 4-methylumbelliferyl ß-d-galactopyranoside are described. The fluorescent product 4-methylumbelliferone is detected simultaneously with the barcode.

Analysis of major bile acids in saliva samples of patients with Barrett's esophagus using high-performance liquid chromatography-electrospray ionization-mass spectrometry.

A fast, non-invasive, high-performance liquid chromatographic screening method with electrospray ionization mass spectrometric detection was developed for the analysis of three major glycine-conjugated bile acids in human saliva. Using a mobile phase composed of 80% methanol and 0.1% formic acid, glycocholic, glycodeoxycholic, and glycochenodeoxycholic acids were separated in less than 4 minutes with sensitivity in the low nM range. Bile acids are thought to contribute to the pathology of various complications in gastroesophageal reflux disease, for instance, Barrett's esophagus, which may eventually lead to esophageal carcinoma. In this pilot study, samples of saliva obtained from 15 patients with Barrett's esophagus of various severities were compared to saliva samples from 10 healthy volunteers. Glycochenodeoxycholic acid was significantly elevated in the patients and principal component analysis of all bile acids could distinguish the most severe Barrett's esophagus patients. We also reported on the detection of glycochenodeoxycholic acid in exhaled breath condensate for the first time. The promising results of this pilot study warrant future investigation, aiming at non-invasive diagnostics of Barrett's esophagus susceptibility in patients with gastroesophageal reflux disease.

Skin wipe test: A simple, inexpensive, and fast approach in the diagnosis of cystic fibrosis.

OBJECTIVE: To assess the performance of a newly developed skin wipe test (SWT) for the diagnosis of cystic fibrosis (CF). STUDY DESIGN: Spontaneously formed sweat from the forearm was wiped by a cotton swab moistened with 100 µL of deionized (DI) water and extracted into 400 µL of DI water (SWT). The conventional Macroduct sweat test (ST) was performed simultaneously. SWT samples of 114 CF patients, 76 healthy carriers, and 58 controls were analyzed by capillary electrophoresis with contactless conductivity detection and Cl- /K+ and (Cl- + Na+ )/K+ ion ratios were evaluated. Chloride concentrations from Macroduct ST were analyzed coulometrically. RESULTS: Analysis of 248 SWT samples and simultaneous Macroduct ST samples showed comparable method performance. Two ion ratios, Cl- /K+ and (Cl- + Na+ )/K+ , from the SWT samples and Cl- values from the ST samples were evaluated to diagnose CF. Sensitivity of the SWT method using the Cl- /K+ ratio (cutoff value 3.9) was 93.9%, compared to 99.1% when using the (Cl- + Na+ )/K+ ratio (cutoff value 5.0) and 98.3% in using Macroduct Cl- (cutoff value higher or equal to 60 mmol/L). The methods' specificities were 97.8%, 94.0%, and 100.0%, respectively. CONCLUSIONS: The developed SWT method with capillary electrophoretic analysis for CF diagnosis performs comparably to the conventional Macroduct ST. The SWT method is simple, fast, inexpensive, and completely noninvasive. Use of an ion ratio in obtained SWT samples is proposed as a new diagnostic parameter that shows significant promise in CF diagnostics.