Small Volumes Of Biological Samples Research Articles

Cryopreservation of organoids: Strategies, innovation, and future prospects.

Organoid technology has demonstrated unique advantages in multidisciplinary fields such as disease research, tumor drug sensitivity, clinical immunity, drug toxicology, and regenerative medicine. It will become the most promising research tool in translational research. However, the long preparation time of organoids and the lack of high-quality cryopreservation methods limit the further application of organoids. Although the high-quality cryopreservation of small-volume biological samples such as cells and embryos has been successfully achieved, the existing cryopreservation methods for organoids still face many bottlenecks. In recent years, with the development of materials science, cryobiology, and interdisciplinary research, many new materials and methods have been applied to cryopreservation. Several new cryopreservation methods have emerged, such as cryoprotectants (CPAs) of natural origin, ice-controlled biomaterials, and rapid rewarming methods. The introduction of these technologies has expanded the research scope of cryopreservation of organoids, provided new approaches and methods for cryopreservation of organoids, and is expected to break through the current technical bottleneck of cryopreservation of organoids. This paper reviews the progress of cryopreservation of organoids in recent years from three aspects: damage factors of cryopreservation of organoids, new protective agents and loading methods, and new technologies of cryopreservation and rewarming.

Dean-Flow-Coupled Elasto-Inertial Focusing Accelerates Exosome Purification to Facilitate Single Vesicle Profiling.

Exosomes are recognized as noteworthy biomarkers playing unprecedented roles in intercellular communication and disease diagnosis and treatment. It is a prerequisite to obtain high-purity exosomes for the comprehension of exosome biochemistry and further illustration of their functionality/mechanisms. However, the isolation of nanoscale exosomes from endogenous proteins is particularly challenging for small-volume biological samples. Herein, a Dean-flow-coupled elasto-inertial microfluidic chip (DEIC) was developed. It consists of a spiral microchannel with dimensional confined concave structures and facilitates elasto-inertial separation of exosomes with lower protein contaminants from cell culture medium and human serum. The presence of 0.15% (w/v) poly-(oxyethylene) controls the elastic lift force acting on suspended nanoscale particles and makes it feasible for field-free purification of integrity exosomes with a 70.6% recovery and a 91.4% removal rate for proteins. As a proof of concept, the technique demonstrated the individual-vesicle-level biomarker (EpCAM and PD-L1) profiling in combination with simultaneous aptamer-mediated analysis to disclose the sensibility for immune response. Overall, DEIC enables the collection of high-purity exosomes and exhibits potential in integration with downstream analyses of exosomes.

Ultrasonic spectroscopy of sessile droplets coupled to optomechanical sensors.

We describe a system for interrogating the acoustic properties of sub-nanoliter liquid samples within an open microfluidics platform. Sessile droplets were deposited onto integrated optomechanical sensors, which possess ambient-medium-noise-limited sensitivity and can thus passively sense the thermally driven acoustic spectrum of the droplets. The droplet acoustic breathing modes manifest as resonant features in the thermomechanical noise spectrum of the sensor, in some cases hybridized with the sensor's own vibrational modes. Excellent agreement is found between experimental observations and theoretical predictions, over the entire ∼0-40 MHz operating range of our sensors. As an application example, we used the technique to monitor the temporal evolution of evaporating droplets. With suitable control over droplet size and morphology, this technique has the potential for precision acoustic sensing of small-volume biological and chemical samples.

High-sensitivity microvolume UV absorption spectrometry for routine analysis of small-volume biological samples.

Substantial improvement of microvolume UV absorption spectrometry in sensitivity, robustness and ease of operation was achieved for routine biological applications. A unique microtubing-based absorption cell (208μm internal diameter) featuring enhanced light transmission with a liquid core waveguide technique provided dramatically enhanced absorption sensitivity, proportional to the extended path length (50mm, from the typical 1mm), whilerobust measurement performance was attained by implementation of preventive measures against bubble trapping along the light path. For pBR322 plasmid DNA, absorbance at 260nm wasreliably measurable down to 0.1ng/μl with repeatability typically 2-3% RSD. The detection limit was 0.03ng/μldsDNA, far lower than the current state-of-the-art∼1ng/μl. Sample consumption for each measurement was 2.4μl. Automated operation implemented for the first time in microvolume spectrophotometry facilitated the ease in handling with small-volume biological samples.

Development of a NanoLC-MS workflow for high-sensitivity global lipidomic analysis

Lipidomics focuses on the comprehensive analysis of lipids and their interactions with other molecules. Many biological samples are available in small volumes or limited amounts and display very complex lipid compositions; thus, highly sensitive lipidomic analysis methods are often needed. NanoLC-MS offers extremely high sensitivity, although it is known to be technically more challenging than the conventional LC-MS approach, requiring greater care and maintenance. This work describes the development and optimization of a nanoLC-MS method for routine analysis of the lipidome of small volumes of biological samples. We focused on achieving robust conditions for high sensitivity analysis of complex samples. The nanoLC method, mass spectrometry conditions and sample preparation by liquid-liquid extraction of lipids were fully optimized using serum samples and deuterated lipid standards, including an evaluation of contamination sources. The performance of the method was assessed through the analysis of human and pig sera, as well as cerebrospinal fluid samples from pigs. This method allowed the detection of 9900 to 12,200 features by employing only 1.0–2.5 μL of serum samples and identification of 5842 lipids from 36 subclasses within a 50-min gradient. The method can be easily adapted to other types of biological samples where only limited volumes are available.

A 3D-printed modularized purification system for rapid, high-throughput MALDI-MS analysis of small-volume biological samples.

We report a modularized sample purification system (MSPS) fabricated by 3D printing for rapid and high-throughput MALDI-MS analysis of various small-volume biological samples. The MSPS presents distinct advantages such as customizability, portability, expandability, simple operation and high throughput and also keeps the flexibility of being customized with more functional modules in future applications.

Probe technique-based generalized multivariate standard addition strategy for the analysis of fluorescence signals with matrix effects

The presence of practically unavoidable matrix effects from the spectroscopically inactive compounds (which may enhance or suppress intensities of spectral measurements of samples) and background interference effects from the spectroscopically active compounds other than the analytes of interest in real-world complex samples can significantly affect the accuracy of quantitative results of optical chem/biosensing techniques, and hence greatly hinder their applications to real-world complex samples. In this contribution, a probe technique-based generalized multivariate standard addition strategy was designed for the analysis of fluorescence signals with both matrix effects and background interference effects. The performance of the proposed strategy was tested on a proof-of-concept system of quantifying Zn2+ in real-world samples such as rice and zinc gluconate oral solution using a selective fluorescence probe dansylaminoethyl-cyclen and compared with the classical univariate standard addition method. Experimental results demonstrated that one standard addition sample was enough for the proposed strategy to provide satisfactory quantitative results (consistent with the results of ICP-MS) for Zn2+ in the real test samples, which were by far better than the corresponding results of the classical univariate standard addition method. The main contributions of this research are 1) to greatly improve the accuracy of quantitative results of optical chem/biosensing techniques, and 2) to open up an avenue for quantitative analysis of small-volume biological samples or some precious samples using invasive techniques in fields such as biomedical sciences, clinical diagnosis and archeology, etc.

Enzymatic method of urea determination in LTCC microfluidic system based on absorption photometry

In this paper a novel microanalytical system based on absorption photometry for urea determination in small-volume biological samples is described. The developed microsystem with built-in microreactor with urease immobilised on its surface was fabricated using low temperature co-fired ceramics (LTCC) technology. For detection of the ammonium ions – product of urea hydrolysis, the modified Berthelot’s reaction was applied and conditions of this reaction were optimised in a way to be suitable for analysis in microsystems and for particular application, with special regards to very small alternation of urea concentration in culture medium. The best results were obtained for reagent R1 composed of: 180 mM sodium salicylate, 15 mM sodium nitroprusside, 2 mM EDTA in phosphate buffer solution of pH 7 and reagent R2 containing 16.9 mM sodium hypochlorite in phosphate buffer solution of pH 12.Linear concentration range of urea detection with the use of the developed LTCC microfluidic system for the selected flow rate of 4 μl/min, was in the range up to 1 mM, and the calculated lower limit of detection – 0.002 mM. The developed microsystem enables urea determination in samples of post-culture medium and of cells lysates of minimised volume c.a. 1 μl. Preliminary studies related to determination of urea in real samples, performed using hepatic cells lysates and post-culture medium were successful. As reference methods, commercially available tests were applied. The determined urea concentration measured by means of the QuantiChrom™ and BioMaxima test and by the developed method based on the LTCC microanalytical system differs by c.a. 9% and 2% respectively. To the best of our knowledge, this is the first urea analysis microsystem for use in cells culture studies characterised by: the smallest volume of sample – 1 μl, long lifetime with stable response - reduced by 14%, through experimental time span of 30 days as well as suitability for quasi-monitoring of cells in vitro with sampling rate - 6 samples per hour.

Application of solid-phase microextraction in current biomedical research.

Extraction of endogenous compounds and drugs and their corresponding metabolites from complex matrices, such as biofluids and solid tissues, requires adequate analytical approach facilitating qualitative and quantitative analysis. To this end, solid-phase microextraction has been introduced as modern technology that is capable of efficient and high-throughput extraction of compounds due to its ability to amalgamate sampling, extraction, and pre-concentration steps, while requiring minimal use of organic solvents. The ability of solid-phase microextraction to enable analyses on small-volume biological samples and growing availability of biocompatible solid-phase microextraction coatings make it a highly useful technology for variety of applications. For example, solid-phase microextraction is particularly useful for identifying biomarkers in metabolomics studies, and it can be successfully applied in pharmaceutical and toxicological studies requiring the fast and sensitive determination of drug levels, especially those that are present at low levels in biological matrices such as plasma, urine, saliva, and hair. Moreover, solid-phase microextraction can be directly applied in in vivo studies because this extraction technique is non-exhaustive and its biocompatible probes offer minimal invasiveness to the analyzed system. In this article, we review recent progress in well-established solid-phase microextraction technique for in vitro and in vivo analyses of various metabolites and drugs in clinical, pharmaceutical, and toxicological applications.

γ-Aminobutyric acid quantification in small volume biological samples through enzymatically induced electrochemiluminescence.

γ-Aminobutyric acid (GABA) is a well-known neurotransmitter that regulates inhibitory neurotransmission in the mammalian central nervous system and participates in several processes outside the brain. A reliable quantification method is needed to determine its role in different physiological and pathological conditions. However, GABA measurements have several challenges because GABA is neither fluorescent nor electroactive, and it is difficult to detect using enzymatic reactions because no oxidases or dehydrogenases have been identified. Several methods have been developed to quantify GABA concentrations based on the instrumentation available, the sensitivity required, and the volume of samples analyzed. Most of these methods use high-performance liquid chromatography (HPLC). Here, we describe a method for quantifying GABA concentrations in small volume samples using enzymatically-induced electrochemiluminescence with the well-known GABAse complex, which produces glutamate for use in a luminescent reaction with glutamate oxidase and luminol in an electrochemiluminescence cell. The luminescence obtained was proportional to the GABA concentrations in the micromolar range (1-1000), with linear r2 values > 0.95. GABA standards were treated with the enzymatic reactors to generate glutamate (Glu), which was measured simultaneously with an HPLC technique, to validate this new procedure. The assay was further used to determine GABA concentrations in hippocampal extracts. This alternative may be used to quantify GABA levels in fluid samples, such as microdialysates, other perfusates and tissue extracts. Thus, the method presented here is a good alternative for monitoring GABA levels with good sensitivity compared with the traditional methods that are still in use.

Simultaneous determination of cis-permethrin and trans-permethrin in rat plasma and brain tissue using gas chromatography-negative chemical ionization mass spectrometry.

A sensitive method for the simultaneous determination of cis-permethrin (cis-PERM) and trans-permethrin (trans-PERM) in small volumes (100μL) of rat plasma and brain homogenate was developed, using a liquid-liquid extraction for sample preparation and gas chromatography-negative chemical ionization mass spectrometry (GCNCI-MS) for detection. Quantitation of trace levels of the insecticide in small volumes of biological samples is essential to support toxicokinetic studies in small animals. There are currently no validated methods in the literature for determining cis-PERM and trans- PERM in volumes as low as 100μL of rat plasma or brain homogenate. The method provided a linear range of 0.2-150.0ng/mL for analytes in both matrices. The intra- and inter-batch precision (as% relative standard deviation, RSD) and accuracy (as relative error, RE) of the method were better than 20% at the limit of quantitation and better than 15% across the remaining linear range. The validated method was applied in a toxicokinetic study in adult rats with oral dosing of 10mg/kg (cis-PERM) and 100mg/kg (trans-PERM) in corn oil. cis-PERM and trans- PERM were monitored in rat plasma and brain tissue samples for 6h following dosing, and both analytes were detected in all plasma and brain samples.

Label‐free surface‐enhanced Raman spectroscopy of lipid‐rafts from hepatocyte plasma membranes

Lipid rafts are sphingomyelin/cholesterol‐rich domains present in the plasma membrane of eukaryotic cells. In the hepatocyte, it has been shown that these domains intervene and modify a wide range of functions from which cell signaling in health and disease is of major importance. The present investigation proposes a novel strategy for the study of plasma membrane lipid rafts using surface‐enhanced Raman spectroscopy (SERS) and spectra processing employing the principal component analysis (PCA) in correlation with conventional biochemical techniques. SERS has been used for artificial membranes; our approach has the advantage that allows studying purified biological membranes using small volumes of biological samples and a very simple protocol. Therefore, the use of SERS/PCA represents an important advantage for the study not only of the structure and composition of biological membranes but also for the understanding of the functions carried by the membrane‐embedded proteins. Our work using SERS/PCA for the first time shows a correlation when studying the composition of lipid rafts from the cell plasma membrane and the catalytic activity of the Ca2+‐ATPase, and its direct association with the presence of specific lipids located in membrane lipid rafts. Also, we show that changes in plasma membrane and the catalytic activity of the Ca2+‐ATPase activity present in the plasma membrane of hepatocytes after partial depletion of membrane cholesterol carried out by methyl‐beta‐cyclodextrin correlate with the changes in the enzyme activity and the presence of cholesterol‐rich domains of lipid rafts. Copyright © 2017 John Wiley & Sons, Ltd.

Rapid Identification of Unknown Organic Iodine in Small-Volume Complex Biological Samples Based on Nanospray Mass Spectrometry Coupled with in-Tube Solid Phase Microextraction.

A new method for rapid screening of unknown organic iodine (OI) in small-volume complex biological samples was developed using in-tube solid phase microextraction (SPME) nanospray mass spectrometry (MS). The method proposed a new identification scheme for OI based on nanospray high-resolution mass spectrometry (HR-MS). The mass ranges of OI ions were confirmed using the t-MS2 scan mode first; then, the possible precursor ions of OI were selected and identified orderly in full MS/ddMS2 and t-MS2 scan modes. Besides, in-tube SPME was used for the pretreatment of small-volume biological samples, and it was the first time in-tube SPME combined with nanospray MS for OI identification. The whole analysis procedure took only 8 min and consumed 50 μL per sample. Using the new method, six kinds of OI added to urine and an unknown OI C12H23O11I in human milk were successfully identified. Moreover, the proposed identification scheme is also suitable for other ambient mass spectrometry (AMS) to determine unknown compounds with characteristic fragment ions.

Direct determination of zinc in plasma by graphite furnace atomic absorption spectrometry using palladium/magnesium and EDTA matrix modification with high temperature pyrolysis

A new graphite furnace atomic absorption spectrometry method is presented for the analysis of zinc in small volume biological samples.

Separating Putative Pathogens from Background Contamination with Principal Orthogonal Decomposition: Evidence for Leptospira in the Ugandan Neonatal Septisome.

Neonatal sepsis (NS) is responsible for over 1 million yearly deaths worldwide. In the developing world, NS is often treated without an identified microbial pathogen. Amplicon sequencing of the bacterial 16S rRNA gene can be used to identify organisms that are difficult to detect by routine microbiological methods. However, contaminating bacteria are ubiquitous in both hospital settings and research reagents and must be accounted for to make effective use of these data. In this study, we sequenced the bacterial 16S rRNA gene obtained from blood and cerebrospinal fluid (CSF) of 80 neonates presenting with NS to the Mbarara Regional Hospital in Uganda. Assuming that patterns of background contamination would be independent of pathogenic microorganism DNA, we applied a novel quantitative approach using principal orthogonal decomposition to separate background contamination from potential pathogens in sequencing data. We designed our quantitative approach contrasting blood, CSF, and control specimens and employed a variety of statistical random matrix bootstrap hypotheses to estimate statistical significance. These analyses demonstrate that Leptospira appears present in some infants presenting within 48 h of birth, indicative of infection in utero, and up to 28 days of age, suggesting environmental exposure. This organism cannot be cultured in routine bacteriological settings and is enzootic in the cattle that often live in close proximity to the rural peoples of western Uganda. Our findings demonstrate that statistical approaches to remove background organisms common in 16S sequence data can reveal putative pathogens in small volume biological samples from newborns. This computational analysis thus reveals an important medical finding that has the potential to alter therapy and prevention efforts in a critically ill population.

Ambient Ionization Mass Spectrometry for Point-of-Care Diagnostics and Other Clinical Measurements.

One driving motivation in the development of point-of-care (POC) diagnostics is to conveniently and immediately provide information upon which healthcare decisions can be based, while the patient is on site. Ambient ionization mass spectrometry (MS) allows direct chemical analysis of unmodified and complex biological samples. This suite of ionization techniques was introduced a decade ago and now includes a number of techniques, all seeking to minimize or eliminate sample preparation. Such approaches provide new opportunities for POC diagnostics and rapid measurements of exogenous and endogenous molecules (e.g., drugs, proteins, hormones) in small volumes of biological samples, especially when coupled with miniature mass spectrometers. Ambient MS-based techniques are applied in diverse fields such as forensics, pharmaceutical development, reaction monitoring, and food analysis. Clinical applications of ambient MS are at an early stage but show promise for POC diagnostics. This review provides a brief overview of various ambient ionization techniques providing background, examples of applications, and the current state of translation to clinical practice. The primary focus is on paper spray (PS) ionization, which allows quantification of analytes in complex biofluids. Current developments in the miniaturization of mass spectrometers are discussed. Ambient ionization MS is an emerging technology in analytical and clinical chemistry. With appropriate MS instrumentation and user-friendly interfaces for automated analysis, ambient ionization techniques can provide quantitative POC measurements. Most significantly, the implementation of PS could improve the quality and lower the cost of POC testing in a variety of clinical settings.

Using Antibody Arrays to Measure Protein Abundance and Glycosylation: Considerations for Optimal Performance

Antibody arrays provide a valuable method for obtaining multiple protein measurements from small volumes of biological samples. Antibody arrays can be designed to target not only core protein abundances (relative or absolute abundances, depending on the availability of standards for calibration), but also posttranslational modifications, provided antibodies or other affinity reagents are available to detect them. Glycosylation is a common modification that has important and diverse functions in both normal and disease biology. Significant progress has been made in developing methods for measuring glycan levels on multiple specific proteins using antibody arrays and glycan-binding reagents. This unit describes practical approaches for developing, optimizing, and using antibody array assays to determine both protein abundance and glycosylation state. Low-volume arrays can be used to reduce sample consumption, and a new way to improve the binding strength of particular glycan-binding reagents through multimerization is discussed. These methods can be useful for a wide range of biological studies in which glycosylation may change and/or affect protein function.

Highly Selective Electrochemical Strategy for Monitoring of Cerebral Cu2+ Based on a Carbon Dot-TPEA Hybridized Surface

Direct determination of cerebral metal ions in small volume biological samples is still the bottleneck for evaluating the roles that metal ions play in the physiological and pathological processes. In this work, selected copper ion (Cu(2+)) as a model, a facile and direct electrochemical method for detection of Cu(2+) has been developed on the basis of two new designed strategies: one is specific recognition molecule for Cu(2+)-AE-TPEA (N-(2-aminoethyl)-N,N',N'-tris(pyridine-2-yl-methyl)ethane-1,2-diamine); another is carbon dots (C-Dots) with high electrocatalytic activity. Based on the high affinity between TPEA and Cu(2+), the electrode assembled with C-Dot-TPEA hybridized nanocomposites shows high selectivity toward Cu(2+) over other metal ions, amino acids, and biological coexisting species, such as uric acid (UA), ascorbic acid (AA), and so on, which makes it possible to be used for determination of Cu(2+) in the complex brain system. By taking advantage of C-Dots, a dynamic linear range from 1 μM to 60 μM is first achieved with a detection limit of ∼100 nM in aCSF solution. In addition, the developed method with theoretical simplicity and less instrumental demands exhibits long-term stability and good reproducibility. As a result, the present strategy has been successfully applied in detection of cerebral Cu(2+) in normal rat brain and that followed by global cerebral ischemia, combined with in vivo microdialysis. The determined concentrations of Cu(2+) in the rat brain microdialysates by the present method are found to be identical to those obtained by the conventional ICP-AES method.

Covalent Conjugation of Antibodies to Biomolecular-Motor Driven Shuttles

Emerging diagnostic needs for the detection of wide arrays of biomarker molecules require the detection of ultralow concentrations of protein in small volumes of biological samples. In past work, a number of microfluidic devices have been developed in attempt to satisfy these needs. One technology incorporates a critical component of the cell's cytoskeletal system, kinesin and microtubules (MT), in engineered device structures to actively collect and concentrate functionalized microtubules into a small concentrator region. The biomolecular motor kinesin hydrolyzes ATP and converts this chemical energy into mechanical work by moving along microtubule filaments. Incorporating MTs coated with antibodies (Ab) into this concentrator device will allow for a large variety of analytes to be captured with high specificity and localized for detection. We use a heterobifunctional, 8.3A long crosslinker to covalently attach a reduced thiol group on the heavy chain of the antibody to a free succinimidyl ester on the outer surface of the microtubule. This conjugation method prevents undesired cross-linked products (MT-MT, Ab-Ab) and is applicable to monoclonal, polyclonal and F(ab)’2 antibodies, specifically anti-BSA and anti-TNFα. The motility of the functionalized microtubule complex was not significantly impaired as the gliding velocity of the functionalized microtubules driven by Kinesin-1 from Neurospora crassa decreased by about 25%, lowering the average velocity of the complex from 2.28 μm/s to 1.71 μm/s. Importantly, image analysis of fluorescent antigens attached to functionalized MTs show that microtubules are conjugated with >100 antibodies/μm of length. Based on these results, we conclude that the high density of antibodies on the microtubules along with the high binding affinity of antigen suggests that these molecular shuttles coupled with fluorescent detection can be incorporated into concentrator devices to detect ultralow concentrations of specific biomolecules.

Liquid chromatography electrospray ionization tandem mass spectrometry analysis method for simultaneous detection of trichloroacetic acid, dichloroacetic acid, S-(1,2-dichlorovinyl)glutathione and S-(1,2-dichlorovinyl)-L-cysteine

Trichloroethylene (TCE, CAS 79-01-6) is a widely used industrial chemical, and a common environmental pollutant. TCE is a well-known carcinogen in rodents and is classified as “probably carcinogenic to humans”. Several analytical methods have been proposed for detection of TCE metabolites in biological media utilizing derivatization-free techniques; however, none of them is suitable for simultaneous detection of both oxidative metabolites and glutathione conjugates of TCE in small volume biological samples. Here, we report a new combination of methods for assessment of major TCE metabolites: dichloroacetic acid (DCA), trichloroacetic acid (TCA), S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and S-(1,2-dichlorovinyl) glutathione (DCVG). First, DCA and TCA were extracted with ether. Second, the remaining aqueous fraction underwent solid phase extraction for DCVC and DCVG. Then, DCA and TCA were measured by hydrophilic interaction liquid chromatography ion exchange negative electrospray ionization tandem mass spectrometry, while DCVC and DCVG were measured by reverse phase positive electrospray ionization tandem mass spectrometry. This method was applied successfully to measure all 4 TCE metabolites in as little as 50 μl of serum from mice orally exposed to TCE (2100 mg/kg, 2 h). Serum concentrations (mean ± standard deviation) of the TCE metabolites obtained with this method are comparable or equivalent to those previously reported in the literature: DCA, 0.122 ± 0.014 nmol/ml (limit of detection: 0.01 nmol/ml); TCA, 256 ± 30 nmol/ml (0.4 nmol/ml); DCVG, 0.037 ± 0.015 nmol/ml (0.001 nmol/ml); DCVC, 0.0024 ± 0.0009 nmol/ml (0.001 nmol/ml). This method opens new opportunities to increase throughput and decrease number of animals required for mechanistic studies on TCE in rodents.