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.

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.

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.

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.

Optimization of background electrolyte composition for simultaneous contactless conductivity and fluorescence detection in capillary electrophoresis of biological samples.

In this article, optimization of BGE for simultaneous separation of inorganic ions, organic acids, and glutathione using dual C4 D-LIF detection in capillary electrophoresis is presented. The optimized BGE consisted of 30 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid, 15 mM 2-amino-2-hydroxymethyl-propane-1,3-diol, and 2 mM 18-crown-6 at pH 7.2 and allowed simultaneous separation of ten inorganic anions and cations, three organic acids and glutathione in 20 min. The samples were injected hydrodynamically from both capillary ends using the double-opposite end injection principle. Sensitive detection of anions, cations, and organic acids with micromolar LODs using C4 D and simultaneously glutathione with nanomolar LODs using LIF was achieved in a single run. The developed BGE may be useful in analyses of biological samples containing analytes with differing concentrations of several orders of magnitude that is not possible with single detection mode.

Upconversion nanoparticle bioconjugates characterized by capillary electrophoresis.

Upconversion nanoparticles (UCNPs) are an emerging class of optical materials with high potential in bioimaging due to practically no background signal and high penetration depth. Their excellent optical properties and easy surface functionalization make them perfect for conjugation with targeting ligands. In this work, capillary electrophoretic (CE) method with laser-induced fluorescence detection was used to investigate the behavior of carboxyl-silica-coated UCNPs. Folic acid, targeting folate receptor overexpressed by wide variety of cancer cells, was used for illustrative purposes and assessed by CE under optimized conditions. Peptide-mediated bioconjugation of antibodies to UCNPs was also investigated. Despite the numerous advantages of CE, this is the first time that CE was employed for characterization of UCNPs and their bioconjugates. The separation conditions were optimized including the background electrolyte concentration and pH. The optimized electrolyte was 20 mM borate buffer with pH 8.

Non-aqueous capillary electrophoretic separation of cholesterol and 25-hydroxycholesterol after derivatization with Girard P reagent.

Capillary electrophoresis (CE) can provide high separation efficiency with very simple instrumentation, but has yet to be explored regarding oxysterols/cholesterol. Cholesterol and 25-hydroxycholesterol (both are 4-ene-3-ketosteroids) were quantitatively transformed into hydrazones using Girard P reagent after enzymatic oxidation by cholesterol oxidase. Separation was achieved using non-aqueous capillary electrophoresis with UV detection at 280nm; the "charge-tagging" Girard P reagent ensured both charge and chromophore (which are requirements for CE-UV). Excess reagent was also separated from the two analytes, eliminating the need for removal prior to the analysis. The compounds were separated in less than 5min with excellent separation efficiency, using separation electrolytes fully compatible with mass spectrometry. The CE-UV method was used to optimize steps for charge-tagging, revealing that the procedure is affected by the analyte/reagent ratio and reaction time, but also the analyte structure.

New approach for cystic fibrosis diagnosis based on chloride/potassium ratio analyzed in non-invasively obtained skin-wipe sweat samples by capillary electrophoresis with contactless conductometric detection.

A new approach for sweat analysis used in cystic fibrosis (CF) diagnosis is proposed. It consists of a noninvasive skin-wipe sampling followed by analysis of target ions using capillary electrophoresis with contactless conductometric detection (C4D). The skin-wipe sampling consists of wiping a defined skin area with precleaned cotton swab moistened with 100 µL deionized water. The skin-wipe sample is then extracted for 3 min into 400 µL deionized water, and the extract is analyzed directly. The developed sampling method is cheap, simple, fast, and painless, and can replace the conventional pilocarpine-induced sweat chloride test commonly applied in CF diagnosis. The aqueous extract of the skin-wipe sample content is analyzed simultaneously by capillary electrophoresis with contactless conductometric detection using a double opposite end injection. A 20 mmol/L L-histidine/2-(N-morpholino)ethanesulfonic acid and 2 mmol/L 18-crown-6 at pH 6 electrolyte can separate all the major ions in less than 7 min. Skin-wipe sample extracts from 30 study participants-ten adult patients with CF (25-50 years old), ten pediatric patients with CF (1-15 years old), and ten healthy control individuals (1-18 years old)-were obtained and analyzed. From the analyzed ions in all samples, a significant difference between chloride and potassium concentrations was found in the CF patients and healthy controls. We propose the use of the Cl-/K+ ratio rather than the absolute Cl- concentration and a cutoff value of 4 in skin-wipe sample extracts as an alternative to the conventional sweat chloride analysis. The proposed Cl-/K+ ion ratio proved to be a more reliable indicator, is independent of the patient's age, and allows better differentiation between non-CF individuals and CF patients having intermediate values on the Cl- sweat test. Figure New approach for cystic fibrosis diagnosis based on skin-wipe sampling of forearm and analysis of ionic content (Cl-/K+ ratio) in skin-wipe extracts by capillary electrophoresis with contactless conductometric detection.

Self-aligning subatmospheric hybrid liquid junction electrospray interface for capillary electrophoresis.

We report a construction of a self-aligning subatmospheric hybrid liquid junction electrospray interface for CE eliminating the need for manual adjustment by guiding the capillaries in a microfabricated liquid junction glass chip at a defined angle. Both the ESI and separation capillaries are inserted into the microfabricated part until their ends touch. The distance between the capillary openings is defined by the angle between the capillaries. The microfabricated part contains channels for placement of the capillaries and connection of the external electrode reservoirs. It was fabricated using standard photolithographic/wet chemical etching techniques followed by thermal bonding. The liquid junction is connected to a subatmospheric electrospray chamber inducing the flow inside the ESI needle and helping the ion transport via aerodynamic focusing.

Monitoring the ionic content of exhaled breath condensate in various respiratory diseases by capillary electrophoresis with contactless conductivity detection.

The analysis of an ionic profile of exhaled breath condensate (EBC) by capillary electrophoresis with contactless conductivity detection and double opposite end injection, is demonstrated. A miniature sampler made from a 2 ml syringe and an aluminium cooling cylinder was used for the fast collection of EBC (under one minute). Analysis of the collected EBC was performed in a 60 mM 2-(N-morpholino)ethanesulfonic acid, 60 mM L-histidine background electrolyte with 30 µM cetyltrimethylammonium bromide and 2 mM 18-crown-6 at pH 6, and excellent repeatability of migration times (RSD <1.3% (n = 7)) and peak areas (RSD < 7% (n = 7)) of 14 ions (inorganic anions, cations and organic acids) was obtained. It is demonstrated that the analysis of EBC samples obtained from patients with various respiratory diseases (chronic obstructive pulmonary disease, asthma, pulmonary fibrosis, sarcoidosis, cystic fibrosis) is possible in less than five minutes and the ionic profile can be compared with the group of healthy individuals. The analysis of the ionic profile of EBC samples provides a set of data in which statistically significant differences among the groups of patients could be observed for several clinically relevant anions (nitrite, nitrate, acetate, lactate). The developed collection system and method provides a highly reproducible and fast way of collecting and analyzing EBC, with future applicability in point-of-care diagnostics.

Rapid and simple preparation of thiol-ene emulsion-templated monoliths and their application as enzymatic microreactors.

A novel, rapid and simple method for the preparation of emulsion-templated monoliths in microfluidic channels based on thiol-ene chemistry is presented. The method allows monolith synthesis and anchoring inside thiol-ene microchannels in a single photoinitiated step. Characterization by scanning electron microscopy showed that the methanol-based emulsion templating process resulted in a network of highly interconnected and regular thiol-ene beads anchored solidly inside thiol-ene microchannels. Surface area measurements indicate that the monoliths are macroporous, with no or little micro- or mesopores. As a demonstration, galactose oxidase and peptide-N-glycosidase F (PNGase F) were immobilized at the surface of the synthesized thiol-ene monoliths via two different mechanisms. First, cysteine groups on the protein surface were used for reversible covalent linkage to free thiol functional groups on the monoliths. Second, covalent linkage was achieved via free primary amino groups on the protein surface by means of thiol-ene click chemistry and l-ascorbic acid linkage. Thus prepared galactose oxidase and PNGase F microreactors demonstrated good enzymatic activity in a galactose assay and the deglycosilation of ribonuclease B, respectively.

Sensitive determination of glutathione in biological samples by capillary electrophoresis with green (515 nm) laser-induced fluorescence detection.

A new sensitive capillary electrophoretic method with laser-induced fluorescence (LIF) was developed for quantitation of glutathione (GSH) in biological samples. Eosin-5-maleimide was used to label the GSH molecule and the formed conjugate was separated in a 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid electrolyte at pH 7.0 in less than 3 min. The conjugate was detected with an in-house built LIF system, utilizing an inexpensive 515 nm diode laser module. Studies were performed to optimize the derivatization (the ratio of reagent to analyte, the reaction time, pH, etc.) and separation conditions. Sensitive detection of GSH at concentrations as low as 0.18 nM was obtained. The method was applied in the analysis of biological fluids (exhaled breath condensate, saliva) and was found to be suitable for determination of GSH in these samples at trace levels below 1 nM. To the best of our knowledge, this is the first report on determination of GSH in exhaled breath condensate by capillary electrophoresis (CE).

Double opposite end injection capillary electrophoresis with contactless conductometric detection for simultaneous determination of chloride, sodium and potassium in cystic fibrosis diagnosis.

A novel approach for diagnosis of cystic fibrosis is presented. A simple and fast procedure to obtain sweat sample was developed. It consists of repeatedly wiping the skin of the forearm with deionized water moisturized cotton swab and extraction in 1mL of deionized water. Double opposite end injection capillary electrophoresis with contactless conductometric detection is used for the analysis of the extract. Chloride, sodium and potassium as the three target ions that participate in the ion transfer across the cellular membranes, and are affected by CF, are simultaneously determined in approximately 3min in a background electrolyte containing 20mM 2-(N-morpholino)ethanesulfonic acid, 20mM l-histidine and 2mM 18-crown-6. By using the target ion ratios rather than the concentrations of each individual ion combined with principal component analysis, the diagnosis of CF can be made more accurately and greatly reduce the number of false positive or negative results as is often the case when single ion (chloride) is analyzed.

Exhaled breath condensate: determination of non-volatile compounds and their potential for clinical diagnosis and monitoring. A review.

Exhaled breath condensate is a promising, non-invasive, diagnostic sample obtained by condensation of exhaled breath. Starting from a historical perspective of early attempts of breath testing towards the contemporary state-of-the-art breath analysis, this review article focuses mainly on the progress in determination of non-volatile compounds in exhaled breath condensate. The mechanisms by which the aerosols/droplets of non-volatile compounds are formed in the airways are discussed with methodological consequences for sampling. Dilution of respiratory droplets is a major problem for correct clinical interpretation of the measured data and there is an urgent need for standardization of EBC. This applies also for collection instrumentation and therefore various commercial and in-house built devices are described and compared with regard to their design, function and collection parameters. The analytical techniques and methods for determination of non-volatile compounds as potential markers of oxidative stress and lung inflammation are scrutinized with an emphasis on method suitability, sensitivity and appropriateness. The relevance of clinical findings for each group of possible non-volatile markers of selected pulmonary diseases and methodological recommendations with emphasis on interdisciplinary collaboration that is essential for future development into a fully validated clinical diagnostic tool are given.

Bioluminescence determination of active caspase-3 in single apoptotic cells.

Caspase-3 is an executive caspase, in the central position within apoptotic machinery. Apoptosis as a way of programmed cell death is a physiological process that plays an essential role in the development and homeostasis maintenance; moreover, its deregulations are linked to tumor progression or various autoimmune disorders. Therefore, an investigation of apoptosis pathways on the level of individual cells is not only of biological but also medical importance. In this work we report on the development of a high-sensitivity instrumentation and protocol for detection of active caspase-3 in individual mammalian apoptotic cells. The technology is based on the specific cleavage of modified luciferin by caspase-3, an immediate bioluminescence reaction of free luciferin with luciferase followed by emissions of photons and their detection by photomultiplier tube working in the photon counting regime. Three different instrumental arrangements are compared for the determination of caspase-3 in free cells or tissue samples. Thus, in our best miniaturized system the mean amount as low as about 6.5 fg corresponding to 122 000 molecules of caspase-3 can be detected in individual apoptotic mouse leg cells.

Nanoparticle-modified monolithic pipette tips for phosphopeptide enrichment.

We have developed nanoparticle-modified monoliths in pipette tips for selective and efficient enrichment of phosphopeptides. The 5 µL monolithic beds were prepared by UV-initiated polymerization in 200 µL polypropylene pipette tips and either iron oxide or hydroxyapatite nanoparticles were used for monolith modification. Iron oxide nanoparticles were prepared by a co-precipitation method and stabilized by citrate ions. A stable coating of iron oxide nanoparticles on the pore surface of the monolith was obtained via multivalent electrostatic interactions of citrate ions on the surface of nanoparticles with a quaternary amine functionalized poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) monolith. Hydroxyapatite nanoparticles were incorporated into the poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) monolith by simply admixing them in the polymerization mixture followed by in situ polymerization. The nanoparticle-modified monoliths were compared with commercially available titanium dioxide pipette tips. Performance of the developed and commercially available sorbents was demonstrated with the efficient and selective enrichment of phosphopeptides from peptide mixtures of α-casein and ß-casein digests followed by off-line MALDI/MS analysis.