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BACKGROUND: Dietary factors, including high sugar intake, may have adverse effects on male reproduction. Studies of the association between sugar-sweetened beverage (SSB) intake and semen quality have reported inconsistent results. OBJECTIVE: We estimated the effects of SSB consumption on semen quality in a North American preconception cohort study. METHODS: We analyzed baseline data from 690 males (n = 1,247 samples) participating in Pregnancy Study Online (PRESTO) during 2015-2022. Participants aged ≥21 years completed a baseline questionnaire on which they reported information about intake of SSBs, including sodas, energy drinks, sports drinks, and fruit juices. After enrollment, we invited U.S. participants to a semen testing substudy, in which they collected and analyzed two samples using an at-home semen testing kit. We used linear regression models to estimate adjusted percent differences (%D) and 95% confidence intervals (CI) for associations of SSB intake with semen volume, sperm concentration, total sperm count (TSC), motility, and total motile sperm count (TMSC). We used modified Poisson regression models to estimate adjusted risk ratios (RRs) and 95% CIs for the association of SSB intake with World Health Organization semen parameter cut points. RESULTS: Relative to non-consumers of SSBs, those who consumed ≥7 SSBs/week had lower semen volume (%D = -6, 95% CI: -13, 0), sperm concentration (%D = -22, 95% CI: -38, 0), TSC (%D = -22, 95% CI: -38, -2), motility (%D = -4, 95% CI: -10, 2), and TMSC (%D = -25, 95% CI: -43, -2). High SSB consumers also had greater risks of low sperm concentration (≤16 million/mL; RR = 1.89, 95% CI: 1.11, 3.21), low TSC (≤39 million; RR = 1.75, 95% CI: 0.92, 3.33), low motility (≤42%; RR = 1.23, 95% CI: 0.87, 1.75) and low TMSC (≤21 million; RR = 1.95, 95% CI: 1.12, 3.38). Associations were stronger among participants with body mass index ≥ 25 kg/m2 . CONCLUSION: Greater SSB consumption was associated with reduced semen quality in a North American preconception cohort.
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BACKGROUND: The increased demand for decentralized blood sample collection presents numerous operational challenges for diagnostics providers. Sample degradation including sample hemolysis due to time, temperature, and handling between collection and laboratory analysis leads to limited test menus and unreliable results. Here we introduce the lightweight, portable Labcorp TrueSpin™ for rapid point-of-care blood separation using commercially available microvolume blood collection tubes. The TrueSpin is a class I FDA-registered device designed for untrained users. The centrifuge runs on AA batteries and separates a blood sample in 5 minutes. METHODS: Here we describe a series of studies evaluating sample quality and analyte stability in serum samples collected into gel microtubes and processed using the TrueSpin. Hemolysis, residual red blood cell concentration, sample volume, and serum-based chemistry analyte stability were evaluated. RESULTS: No significant difference was seen in hemolysis or residual red blood cell concentration in serum samples prepared by TrueSpin compared to the reference method. Additionally, capillary and venous blood samples separated using the TrueSpin and exposed to International Safe Transit Association 3A-simulated shipping conditions were shown to yield acceptable sample volume and quality for laboratory analysis. Finally, we show that many common serum-based chemistry analytes have limited (< 1 day) stability if uncentrifuged but improve to ≥ 3-day stability following TrueSpin separation and refrigerated or room temperature storage. CONCLUSIONS: These findings suggest that the TrueSpin is a simple and effective solution for remote sample separation and may enable broader test menus and increased test result reliability for decentralized sample collection pursuits.
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Recolección de Muestras de Sangre , Hemólisis , Humanos , Reproducibilidad de los Resultados , Recolección de Muestras de Sangre/métodos , Manejo de Especímenes , Factores de TiempoRESUMEN
OBJECTIVE: To evaluate the association between anthropometric measures and semen parameters. DESIGN: Cross-sectional study. SUBJECTS: Male participants aged ≥21 years. We analyzed data from 659 males (1185 samples) participating in a semen testing substudy of the Pregnancy Online Study (PRESTO), a North American preconception cohort study. After enrollment, we invited males aged ≥21 years to perform at-home semen testing using the Trak system. EXPOSURE(S): Participants reported selected anthropometric variables (current weight, height, waist circumference, and weight at age 17 years) and covariate data via an online baseline questionnaire. MAIN OUTCOME MEASURE(S): We used generalized estimating equations models to estimate the percent difference in mean log-transformed semen parameter values and 95% confidence intervals (CI) for associations between selected anthropometric variables and semen volume (mL), sperm concentration (million/mL), and total sperm count (million), adjusting for sociodemographics, lifestyle factors, and medical history. We also evaluated World Health Organization-defined thresholds for low semen quality. RESULT(S): Percentage differences in mean log-transformed semen volume, sperm concentration, and total sperm count (95% CI) comparing current body mass index ≥35 vs. <25 kg/m2 were -6.3 (-15.8, 4.3), -6.4 (-24.6, 16.2), and -12.2 (-31.1, 11.8), respectively. Percentage differences (95% CIs) comparing waist circumferences of ≥42 vs. <31 inches were -4.2 (-15.0, 8.0), -6.4 (-27.6, 21.0), and -10.4 (-31.9, 17.9) for semen volume, sperm concentration, and total sperm count, respectively. Greater adult weight gain since age 17 years was associated with reduced semen volume (≥25 vs. <5 kg; percent difference, -9.7; 95% CI, -18.4, 0.1), but not sperm concentration or total sperm count. The highest categories of each anthropometric variable generally were associated with World Health Organization-defined low total sperm count (≤39 million). CONCLUSION(S): Selected anthropometric factors were associated modestly with poorer semen quality.
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Análisis de Semen , Semen , Adulto , Embarazo , Femenino , Masculino , Humanos , Adolescente , Estudios de Cohortes , Estudios Transversales , Motilidad Espermática , Recuento de Espermatozoides , Espermatozoides , América del NorteRESUMEN
BACKGROUND: Psychological stress is prevalent among reproductive-aged men. Assessment of semen quality for epidemiological studies is challenging as data collection is expensive and cumbersome, and studies evaluating the effect of perceived stress on semen quality are inconsistent. OBJECTIVE: To examine the association between perceived stress and semen quality. MATERIAL AND METHODS: We analyzed baseline data on 644 men (1,159 semen samples) from two prospective preconception cohort studies during 2015-2021: 592 in Pregnancy Study Online (PRESTO) and 52 in SnartForaeldre.dk (SF). At study entry, men aged ≥21 years (PRESTO) and ≥18 years (SF) trying to conceive without fertility treatment completed a questionnaire on reproductive and medical history, socio-demographics, lifestyle, and the 10-item version of the Perceived Stress Scale (PSS; interquartile range [IQR] of scores: 0-40). After enrollment (median weeks: 2.1, IQR: 1.3-3.7), men were invited to perform in-home semen testing, twice with 7-10 days between tests, using the Trak Male Fertility Testing System. Semen quality was characterized by semen volume, sperm concentration, and total sperm count. We fit generalized estimating equation linear regression models to estimate the percent difference in mean log-transformed semen parameters by four PSS groups (<10, 10-14, 15-19, ≥20), adjusting for potential confounders. RESULTS: The median PSS score and IQR was 15 (10-19), and 136 men (21.1%) had a PSS score ≥20. Comparing men with PSS scores ≥20 with <10, the adjusted percent difference was -2.7 (95% CI: -9.8; 5.0) for semen volume, 6.8 (95% CI: -10.9; 28.1) for sperm concentration, and 4.3 (95% CI: -13.8; 26.2) for total sperm count. CONCLUSION: Our findings indicate that perceived stress is not materially associated with semen volume, sperm concentration, or total sperm count.
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Análisis de Semen , Semen , Embarazo , Femenino , Masculino , Humanos , Adulto , Estudios Prospectivos , Recuento de Espermatozoides , Espermatozoides , Estrés Psicológico , Motilidad EspermáticaRESUMEN
Blood sample collection and rapid separation-critical preanalytical steps in clinical chemistry-can be challenging in decentralized collection settings. To address this gap, the Torq™ zero delay centrifuge system includes a lightweight, hand-portable centrifuge (ZDrive™) and a disc-shaped blood collection device (ZDisc™) enabling immediate sample centrifugation at the point of collection. Here, we report results from clinical validation studies comparing performance of the Torq System with a conventional plasma separation tube (PST). Blood specimens from 134 subjects were collected and processed across three independent sites to compare ZDisc and PST performance in the assessment of 14 analytes (K, Na, Cl, Ca, BUN, creatinine, AST, ALT, ALP, total bilirubin, albumin, total protein, cholesterol, and triglycerides). A 31-subject precision study was performed to evaluate reproducibility of plasma test results from ZDiscs, and plasma quality was assessed by measuring hemolysis and blood cells from 10 subject specimens. The ZDisc successfully collected and processed samples from 134 subjects. ZDisc results agreed with reference PSTs for all 14 analytes with mean % biases well below clinically significant levels. Results were reproducible across different operators and ZDisc production lots, and plasma blood cell counts and hemolysis levels fell well below clinical acceptance thresholds. ZDiscs produce plasma samples equivalent to reference PSTs. Results support the suitability of the Torq System for remotely collecting and processing blood samples in decentralized settings.
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OBJECTIVE: To evaluate the associations of a history of diagnosed depression, current depressive symptoms, and recent use of psychotropic medications with semen quality and to consider mediation of the association between depression and semen quality by medication use. DESIGN: Prospective cohort study. SETTING: United States. PATIENT(S): The patients were 329 men aged ≥21 years (566 semen samples) who participated in a semen-testing substudy of Pregnancy Study Online. Pregnancy Study Online is an ongoing, web-based preconception cohort study of couples attempting to conceive. At baseline, participants reported information about depression diagnosis, depressive symptoms using the Major Depression Inventory, medication use in the last 4 weeks, and selected covariates. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The men used an at-home semen-testing kit (Trak; Sandstone Diagnostics, Inc., Pleasanton, California) to measure semen volume, sperm concentration, and motile sperm concentration. We calculated percent motility, total sperm count in the ejaculate, and total motile sperm count. RESULT(S): Forty-nine men (15%) reported a history of depression diagnosis, and 41 (12%) reported recent use of psychotropic medications. A history of depression diagnosis was associated with a 4.3-fold increase in the risk of low semen volume (<1.5 mL) (95% CI 1.16, 16). A 5-unit increase in Major Depression Inventory score was associated with a 1.38-fold increase in the risk of low semen volume (95% CI 0.92, 2.1). The results for other semen parameters were inconsistent. Recent use of psychotropic medications was associated with worse semen quality, and this association was confounded by a history of depression diagnosis. The observed association between depression and semen volume showed little mediation by psychotropic medication use. CONCLUSION: A history of diagnosed depression and severe depressive symptoms at enrollment were associated with low semen volume.
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Trastorno Depresivo Mayor/complicaciones , Psicotrópicos/uso terapéutico , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Adulto , Trastorno Depresivo Mayor/diagnóstico , Trastorno Depresivo Mayor/patología , Humanos , Masculino , Estudios Prospectivos , Psicotrópicos/efectos adversos , Recuento de Espermatozoides , Espermatozoides/patología , Estados Unidos , Adulto JovenRESUMEN
BACKGROUND: Semen quality assessment in population-based epidemiologic studies presents logistical and financial challenges due to reliance on centralised laboratory semen analysis. The Trak Male Fertility Testing System is an FDA-cleared and validated at-home test for sperm concentration and semen volume, with a research use only sperm motility test. Here we evaluate the Trak System's overall utility among men participating in Pregnancy Study Online (PRESTO), a web-based study of North American couples planning pregnancy. METHODS: US male participants aged ≥21 years with ≤6 months of pregnancy attempt time at study enrolment were invited to participate in the semen testing substudy after completing their baseline questionnaire. Consenting participants received a Trak Engine (battery-powered centrifuge) and two test kits. Participants shared their test results via smartphone images uploaded to online questionnaires. Data were then linked with covariate data from the baseline questionnaire. RESULTS: Of the 688 men invited to participate, 373 (54%) provided consent and 271 (73%) completed at least one semen test result. The distributions of semen volume, sperm concentration, motile sperm concentration, total sperm count, and total motile sperm count were similar to 2010 World Health Organization (WHO) semen parameter data of men in the general population. The overall usability score for the Trak System was 1.4 on a 5-point Likert scale (1 = Very Easy, 5 = Difficult), and 92% of participants believed they performed the test correctly and received an accurate result. Lastly, men with higher motile sperm count were more likely to report feeling "at ease" or "excited" following testing, while men with low motile sperm count were more likely to report feeling "concerned" or "frustrated." Overall, 91% of men reported they would like to test again. CONCLUSIONS: The Trak System provides a simple and potentially cost-effective means of measuring important semen parameters and may be useful in population-based epidemiologic fertility studies.
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Internet , Autoevaluación , Recuento de Espermatozoides/métodos , Motilidad Espermática , Adulto , Estudios Epidemiológicos , Humanos , Infertilidad Masculina/diagnóstico , Masculino , Persona de Mediana Edad , Satisfacción del Paciente , Atención Preconceptiva , Análisis de Semen/instrumentación , Análisis de Semen/métodos , Recuento de Espermatozoides/instrumentación , Adulto JovenRESUMEN
OBJECTIVE: To evaluate the analytical performance and usability of the Trak Male Fertility Testing System, a semiquantitative (categorical) device recently US Food and Drug Administration (FDA)-cleared for measuring sperm concentration in the home by untrained users. DESIGN: A three-site clinical trial comparing self-reported lay user results versus reference results obtained by computer-aided semen analysis (CASA). SETTING: Simulated home use environments at fertility centers and urologist offices. PATIENT(S): A total of 239 untrained users. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Sperm concentration results reported from self-testing lay users and laboratory reference method by CASA were evaluated semiquantitatively against the device's clinical cutoffs of 15 M/mL (current World Health Organization cutoff) and 55 M/mL (associated with faster time to pregnancy). Additional reported metrics include assay linearity, precision, limit of detection, and ease-of-use ratings from lay users. RESULT(S): Lay users achieved an accuracy (versus the reference) of 93.3% (95% confidence interval [CI] 84.1%-97.4%) for results categorized as ≤15 M/mL, 82.4% (95% CI 73.3%-88.9%) for results categorized as 15-55 M/mL, and 95.5% (95% CI 88.9%-98.2%) for results categorized as >55 M/mL. When measured quantitatively, Trak results had a strong linear correlation with CASA measurements (r = 0.99). The precision and limit of detection studies show that the device has adequate reproducibility and detection range for home use. Subjects generally rated the device as easy to use. CONCLUSION(S): The Trak System is an accurate tool for semiquantitatively measuring sperm concentration in the home. The system may enable screening and longitudinal assessment of sperm concentration at home. CLINICAL TRIAL REGISTRATION NUMBER: ClinicalTrials.gov identifier: NCT02475395.
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Centrifugación/instrumentación , Fertilidad , Infertilidad Masculina/diagnóstico , Autocuidado/instrumentación , Recuento de Espermatozoides/instrumentación , Espermatozoides/patología , Adulto , California , Centrifugación/normas , Diseño de Equipo , Humanos , Infertilidad Masculina/etiología , Infertilidad Masculina/patología , Infertilidad Masculina/fisiopatología , Límite de Detección , Modelos Lineales , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Estándares de Referencia , Reproducibilidad de los Resultados , Autocuidado/métodos , Autocuidado/normas , Recuento de Espermatozoides/métodos , Recuento de Espermatozoides/normas , Adulto JovenRESUMEN
We present an innovative centrifugal microfluidic immunoassay platform (SpinDx) to address the urgent biodefense and public health need for ultrasensitive point-of-care/incident detection of botulinum toxin. The simple, sample-to-answer centrifugal microfluidic immunoassay approach is based on binding of toxins to antibody-laden capture particles followed by sedimentation of the particles through a density-media in a microfluidic disk and quantification by laser-induced fluorescence. A blind, head-to-head comparison study of SpinDx versus the gold-standard mouse bioassay demonstrates 100-fold improvement in sensitivity (limit of detection = 0.09 pg/mL), while achieving total sample-to-answer time of <30 min with 2-µL required volume of the unprocessed sample. We further demonstrate quantification of botulinum toxin in both exogeneous (human blood and serum spiked with toxins) and endogeneous (serum from mice intoxicated via oral, intranasal, and intravenous routes) samples. SpinDx can analyze, without any sample preparation, multiple sample types including whole blood, serum, and food. It is readily expandable to additional analytes as the assay reagents (i.e., the capture beads and detection antibodies) are disconnected from the disk architecture and the reader, facilitating rapid development of new assays. SpinDx can also serve as a general-purpose immunoassay platform applicable to diagnosis of other conditions and diseases.
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Toxinas Botulínicas/sangre , Toxinas Botulínicas/química , Inmunoensayo/instrumentación , Microfluídica/instrumentación , Animales , Toxinas Botulínicas/inmunología , Femenino , Análisis de los Alimentos , Humanos , RatonesRESUMEN
In this work, we introduce microscale isoelectric fractionation (µIF) for isolation and enrichment of molecular species at any desired location in a microfluidic chip. Narrow pH-specific polyacrylamide membranes are photopatterned in situ for customizable device fabrication; multiple membranes of precise pH are easily incorporated throughout existing channel layouts. Samples are electrophoretically driven across the membranes such that charged species, for example, proteins and peptides, are rapidly discretized into fractions based on their isoelectric points (pI) without the use of carrier ampholytes. This format makes fractions easy to compartmentalize and access for integrated preparative or analytical operations on-chip. We present and discuss the key design considerations and trade-offs associated with proper system operation and optimal run conditions. Efficient and reproducible fractionation of model fluorescent pI markers and proteins is achieved using single membrane fractionators at pH 6.5 and 5.3 from both buffer and Escherichia coli cell lysate sample conditions. Effective fractionation is also shown using a serial 3-membrane fractionator tailored for isolating analytes-of-interest from high abundance components of serum. We further demonstrate that proteins focused in pH specific bins can be rapidly and efficiently transferred to another location in the same chip without unwanted dilution or dispersive effects. µIF provides a rapid and versatile option for integrated sample prep or multidimensional analysis, and addresses the glaring proteomic need to isolate trace analytes from high-abundance species in minute volumes of complex samples.
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Resinas Acrílicas/química , Membranas Artificiales , Animales , Biomarcadores/análisis , Proteína C-Reactiva/aislamiento & purificación , Anhidrasa Carbónica II/aislamiento & purificación , Anhidrasa Carbónica II/metabolismo , Bovinos , Hemoglobinas/aislamiento & purificación , Humanos , Concentración de Iones de Hidrógeno , Inmunoglobulina G/aislamiento & purificación , Focalización Isoeléctrica , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Ratones , Técnicas Analíticas Microfluídicas , Albúmina Sérica/aislamiento & purificaciónRESUMEN
BACKGROUND: Centrifugal "lab on a disk" microfluidics is a promising avenue for developing portable, low-cost, automated immunoassays. However, the necessity of incorporating multiple wash steps results in complicated designs that increase the time and sample/reagent volumes needed to run assays and raises the probability of errors. We present proof of principle for a disk-based microfluidic immunoassay technique that processes blood samples without conventional wash steps. METHODS: Microfluidic disks were fabricated from layers of patterned, double-sided tape and polymer sheets. Sample was mixed on-disk with assay capture beads and labeling antibodies. Following incubation, the assay beads were physically separated from the blood cells, plasma, and unbound label by centrifugation through a density medium. A signal-laden pellet formed at the periphery of the disk was analyzed to quantify concentration of the target analyte. RESULTS: To demonstrate this technique, the inflammation biomarkers C-reactive protein and interleukin-6 were measured from spiked mouse plasma and human whole blood samples. On-disk processing (mixing, labeling, and separation) facilitated direct assays on 1-µL samples with a 15-min sample-to-answer time, <100 pmol/L limit of detection, and 10% CV. We also used a unique single-channel multiplexing technique based on the sedimentation rate of different size or density bead populations. CONCLUSIONS: This portable microfluidic system is a promising method for rapid, inexpensive, and automated detection of multiple analytes directly from a drop of blood in a point-of-care setting.
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Inmunoensayo/métodos , Microesferas , Animales , Proteína C-Reactiva/análisis , Centrifugación Zonal , Humanos , Inmunoensayo/instrumentación , Interleucina-6/sangre , Ratones , Técnicas Analíticas Microfluídicas , Tamaño de la PartículaRESUMEN
Nucleic acid based affinity reagents (e.g., aptamers) offer several possible advantages over antibodies as specific recognition elements in biochemical assays. Besides offering improved cost and stability, aptamers are ideal for rapid electrophoretic analysis due to their low molecular weight and high negative charge. While aptamers have proven well-suited for affinity-shift electrophoretic analysis, demonstrating a fully integrated aptamer-based assay platform represents an important achievement toward low-cost point-of-care analysis, particularly for remote or resource poor settings where cost and ambient stability of reagents is a key consideration. Here we perform and evaluate the suitability of aptamer-based affinity assays for two clinically relevant target analytes (IgE using a known aptamer and NF-κB using a thio-modified aptamer) in an integrated electrophoretic gel-shift platform. Key steps of (i) mixing sample with aptamer, (ii) buffer exchange, and (iii) preconcentration of sample were successfully integrated on-chip upstream of a fluorescence-based gel-shift analysis step. This approach, utilizing a size-exclusion membrane optimized here for aptamer retention and preconcentration with sample, enables automated sample-to-answer for trace analytes in 10 min or less. We addressed notable nonspecific interference from serum proteins by adding similar nucleic acid competitors to suppress such interactions with the aptamer. Nanomolar sensitivities were demonstrated and integrated preconcentration of sample provides an important means of further improving detection sensitivities. Aptamers proved superior in many respects to antibody reagents, particularly with regard to speed and resolution of gel-shifts associated with specific binding to target.
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Aptámeros de Nucleótidos/química , Inmunoglobulina E/análisis , Dispositivos Laboratorio en un Chip , FN-kappa B/análisis , Secuencia de Bases , Electroforesis/instrumentación , Diseño de Equipo , Humanos , Datos de Secuencia Molecular , FN-kappa B/sangre , Sensibilidad y EspecificidadRESUMEN
In this work we photopolymerized precise and well-controlled polyacrylamide porosity gradients in microchannels for microscale pore limit electrophoresis (microPLE) of proteins. Porosity was controlled via distributions of acrylamide monomer and bisacrylamide crosslinker. MicroPLE provides high-resolution fractionation of complex samples based on the spatial dependence of each species' electrophoretic pore limit--the porosity at which a protein's electrophoretic mobility is negligible due to its molecular size. Proteins ranging in molecular weight from 21.5 kDa-144 kDa were separated under native buffering conditions along 5-mm- and 7-mm-long microPLE gels spanning 10%T, 2.6%C-40%T, 12%C. The pore gradient gel is useful for estimating size-exclusion thresholds for a broad range of polymer concentrations and protein sizes simultaneously. We show that microPLE can be used to concentrate dilute samples by exploiting the stacking phenomenon associated with an analyte's decreasing electrophoretic mobility. Concentration factors>40,000 were demonstrated with dilute (100 pM) samples. A detailed theoretical analysis of microPLE transport behavior based on Ferguson assumptions provides scaling and design parameters with which to tailor gels based on fractionation or enrichment needs. Experimental results show that the Ferguson assumptions break down as proteins migrate beyond an effective pore limit, prompting the need for further investigation into this non-Ferguson regime.
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Electroforesis en Gel de Poliacrilamida/métodos , Proteínas/química , Algoritmos , Electroquímica , Campos Electromagnéticos , Electroforesis en Gel de Poliacrilamida/instrumentación , Diseño de Equipo , Colorantes Fluorescentes , Peso Molecular , PorosidadRESUMEN
IEF is one of the most powerful and prevalent techniques used in separation sciences. The power of IEF comes from the fact that it not only separates analytes based on their pI but also focuses them into highly resolved bands. In line with the miniaturization trend spurring the analytical community, the past decade has yielded a wealth of research focused on implementing IEF in microfluidic chip-based formats (microIEF). Scaling down the separation technique provides several advantages such as reduced sample sizes, assay automation, and significant improvements in assay speed without sacrificing separation performance. Besides presenting microscale adaptations of standard schemes, researchers have also developed improved detection techniques, demonstrated novel microIEF assays, and incorporated microIEF with other analytical methods for achieving on-chip multidimensional separations. This review provides a brief historical outline of IEF's beginnings, theoretical incentives driving miniaturization of the methodology, a thorough synopsis of microIEF publications to date, and an outlook to the future.
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Focalización Isoeléctrica , Técnicas Analíticas Microfluídicas/métodos , Diseño de Equipo , Focalización Isoeléctrica/instrumentación , Focalización Isoeléctrica/métodos , Focalización Isoeléctrica/tendenciasRESUMEN
We present the first successful adaptation of immobilized pH gradients (IPGs) to the microscale (muIPGs) using a new method for generating precisely defined polymer gradients on-chip. Gradients of monomer were established via diffusion along 6 mm flow-restricted channel segments. Precise control over boundary conditions and the resulting gradient is achieved by continuous flow of stock solutions through side channels flanking the gradient segment. Once the desired gradient is established, it is immobilized via photopolymerization. Precise gradient formation was verified with spatial and temporal detection of a fluorescent dye added to one of the flanking streams. Rapid (<20 min) isoelectric focusing of several fluorescent pI markers and proteins is demonstrated across pH 3.8-7.0 muIPGs using both denaturing and nondenaturing conditions, without the addition of carrier ampholytes. The muIPG format yields improved stability and comparable resolution to prominent on-chip IEF techniques. In addition to rapid, high-resolution separations, the reported muIPG format is amenable to multiplexed and multidimensional analysis via custom gradients as well as integration with other on-chip separation methods.
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Focalización Isoeléctrica/métodos , Análisis por Micromatrices/métodos , Proteínas/aislamiento & purificación , Resinas Acrílicas/química , Concentración de Iones de Hidrógeno , Membranas Artificiales , Fotoquímica , Espectrometría de FluorescenciaRESUMEN
We present a detailed theoretical and numerical analysis of temperature gradient focusing (TGF) via Joule heating-an analytical species concentration and separation technique relying upon the dependence of an analyte's velocity on temperature due to the temperature dependence of a buffer's ionic strength and viscosity. The governing transport equations are presented, analyzed, and implemented into a quasi-1D numerical model to predict the resulting temperature, velocity, and concentration profiles along a microchannel of varying width under an applied electric field. Numerical results show good agreement with experimental trials presented in previous work. The model is used to analyze the effects of varying certain geometrical and experimental parameters on the focusing performance of the device. Simulations also help depict the separation capability of the device, as well as the effectiveness of different buffer systems used in the technique. The analysis provides rule-of-thumb methodology for implementation of TGF into analytical systems, as well as a fundamental model applicable to any lab-on-a-chip system in which Joule heating and temperature-dependent electrokinetic transport are to be analyzed.
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Química Física/métodos , Calor , Técnicas Analíticas Microfluídicas , Simulación por Computador , Electroquímica/métodos , Electroósmosis , Cinética , Microfluídica , Modelos Estadísticos , Modelos Teóricos , Electricidad Estática , Temperatura , Termodinámica , Factores de TiempoRESUMEN
We present an experimental study of temperature gradient focusing (TGF) exploiting an inherent Joule heating phenomenon. A simple variable-width PDMS device delivers rapid and repeatable focusing of model analytes using significantly lower power than conventional TGF techniques. High electric potential applied to the device induces a temperature gradient within the microchannel due to the channel's variable width, and the temperature-dependent mobility of the analytes causes focusing at a specific location. The PDMS device also shows simultaneous separation and concentration capability of a mixture of two sample analytes in less than 10 min. An experiment combining Joule heating with external heating/cooling further supports the hypothesis that temperature is indeed the dominant factor in achieving focusing with this technique.