RESUMO
A low cost three-dimensional (3D) printed clear plastic microfluidic device was fabricated for fast, low cost automated protein detection. The unibody device features three reagent reservoirs, an efficient 3D network for passive mixing, and an optically transparent detection chamber housing a glass capture antibody array for measuring chemiluminescence output with a CCD camera. Sandwich type assays were built onto the glass arrays using a multi-labeled detection antibody-polyHRP (HRP = horseradish peroxidase). Total assay time was â¼30 min in a complete automated assay employing a programmable syringe pump so that the protocol required minimal operator intervention. The device was used for multiplexed detection of prostate cancer biomarker proteins prostate specific antigen (PSA) and platelet factor 4 (PF-4). Detection limits of 0.5 pg mL-1 were achieved for these proteins in diluted serum with log dynamic ranges of four orders of magnitude. Good accuracy vs. ELISA was validated by analyzing human serum samples. This prototype device holds good promise for further development as a point-of-care cancer diagnostics tool.
Assuntos
Biomarcadores Tumorais/sangue , Imunoensaio/instrumentação , Imunoensaio/métodos , Medições Luminescentes/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Desenho de Equipamento , Humanos , Limite de Detecção , Masculino , Impressão Tridimensional , Antígeno Prostático Específico/sangue , Neoplasias da PróstataRESUMO
Immunoassays employ antibodies and labels to capture and detect target macromolecular analytes, often from complex sample matrices such as serum, plasma, or saliva. The high affinity and specificity of antibody-antigen interactions makes immunoassays critically important analytical techniques for clinical diagnostics as well as other research applications in the areas of pharmaceutical and environmental analysis. Integration of magnetic beads (MBs) into immunoassays and other bioanalytical methodologies is a valuable approach to allow efficient target capture, enrichment, and convenient separation. In addition, large signal amplification can be achieved by preconcentration of the target and by attaching many thousands of enzyme labels to the MBs. These features have enabled MB-based biosensors to achieve ultra-low detection limits needed for advanced clinical diagnostics that are challenging or impossible using traditional immunoassays. MBs are employed either as mobile substrates for target analyte capture, as detection labels (or label carriers), or simultaneously as substrates and labels. For optimal assay performance, it is crucial to apply an easy, efficient, and robust bead-probe conjugation protocol, and to thoroughly characterize the bioconjugated products. Herein, we describe methods used in our laboratory to functionalize MBs with antibodies and enzyme labels for ultrasensitive detection of protein analytes. We also present detailed strategies for characterizing the MB bioconjugates.
Assuntos
Anticorpos Imobilizados/química , Técnicas Biossensoriais , Enzimas Imobilizadas/química , Marcadores de Afinidade/química , Peroxidase do Rábano Silvestre/química , Imunoensaio , Imãs/química , Técnicas Analíticas Microfluídicas , Nanopartículas/química , Estreptavidina/químicaRESUMO
Inexpensive, reusable electrochemical sensor chips were fabricated from gold CDs. All reagents were loaded onto a paper disk sequentially, then placed on the chip to detect cancer biomarker prostate specific antigen (PSA) in serum at pg mL-1 levels in â¼15 mins.
RESUMO
Cyclic voltammetry of thin films made from the lipid dimyristoylphosphatidyl choline and reaction centers from the purple bacterium Rhodobacter sphaeroides on pyrolytic graphite electrodes in bromide-free pH 8 buffers at 4 degrees C revealed an oxidation peak at 0.98 V and a reduction peak at -0.17 V vs. NHE. No reverse CV peaks were found, suggesting chemical irreversibility. The reduction peak disappeared for reaction centers depleted of quinones, suggesting that the peak represents reduction of this cofactor. The oxidation peak showed a catalytic current increase in the presence of small amounts of ferrous cytochrome c, and decreased by 85% when illuminated by visible light, suggesting assignment to the primary donor (P) cofactor. While oxidized primary donor P(+) is destroyed upon electrochemical formation in the film, reaction of ferrous cyt c with P(+) suggests its persistence in the films on the microsecond time scale.
Assuntos
Complexo de Proteínas do Centro de Reação Fotossintética/química , Quinonas/química , Rhodobacter sphaeroides/química , Grupo dos Citocromos c/farmacologia , Dimiristoilfosfatidilcolina , Eletroquímica , Eletrodos , Grafite , Luz , Membranas Artificiais , Oxirredução , Complexo de Proteínas do Centro de Reação Fotossintética/efeitos da radiaçãoRESUMO
A sensor constructed by alternate layer-by-layer adsorption of PDDA cations and double-stranded (ds)-DNA on oxidized pyrolytic graphite electrodes was evaluated for detection of chemical damage to ds-DNA from known damage agent styrene oxide. Films made with PDDA ions of structure (PDDA/DNA)2 were approximately 6 nm thick and contained 0.23 microg of ds-DNA. Catalytic oxidation using 50 microM Ru(bpy)3(2+) (bpy = 2,2'-bipyridine) and square wave voltammetry (SWV) provided more sensitive detection of DNA damage than direct SWV oxidation. The catalytic peaks increased linearly with time during incubations with styrene oxide, but only minor changes were detected during incubation with nonreactive toluene. For best sensitivity, the outer layer of the film must be ds-DNA, and analysis should be done at low salt concentration. Studies of DNA and polynucleotides in solutions and films suggested that oxidation of guanine and chemically damaged adenine in partly unraveled, damaged DNA were the most likely contributors to the catalytic peak.
Assuntos
2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/química , Dano ao DNA , DNA/análise , Polietilenos/química , Compostos de Amônio Quaternário/química , Animais , Bovinos , Complexos de Coordenação , DNA/química , Eletroquímica/métodos , Eletrodos , Compostos de Epóxi/química , Grafite/química , Oxirredução , Poliaminas/química , PolieletrólitosRESUMO
A strong influence of bromide ion was found on voltammetry of layered films of photosynthetic reaction center (RC) protein and polyions on gold electrodes. Similar, but not identical, cyclic voltammetry peaks were observed for polyion films on gold with and without RC when the buffer solutions contained bromide ion. CVs of RC films were quite different in the absence of bromide. These new findings suggest that previously published results were biased by significant background peaks involving bromide ion adsorption/desorption.
Assuntos
Brometos/química , Eletrodos , Ouro , FotossínteseRESUMO
Quartz crystal microbalance (QCM) resonance measurements were used to examine the surface charge characteristics of cytochrome P450 forms and the influence of charge on the docking of redox partners like cytochrome b5. The distal surface of cytochrome P450 (CYP)101 (pI = 4.5), relative to the heme, is fairly anionic, as is the proximal surface. The latter, however, also has two cationic clusters. A considerably greater extent of CYP101 binding was seen to the cationic, polyethylene-surfaced resonators. CYP2B4 (pI = 8.5) preferentially bound to the polyanionic, polystyrene sulfonate-surfaced resonators. Cytochrome b5 is an acidic protein that had a preferential binding to the poly(ethyleneimine (PEI)-surfaced resonators. When binding to CYP2B4-surfaced films, cytochrome b5 preferentially bound to those cytochrome P450 molecules that were adsorbed to cationic (PEI) films. It is suggested that adsorption of CYP2B4 to an anionic poly(styrenesulfonate) (PSS) surface is with cationic clusters that include the cytochrome b5 docking domain. This diminishes the extent of docking of the cytochrome b5. In contrast, when CYP2B4 is adsorbed to a cationic film the proximal surface with the cytochrome b5-docking site is available for cytochrome b5 binding. A film of the polycation PEI was adsorbed to the silver QCM surface. It formed polymer islands when viewed with atomic force microscopy. Polyanionic PSS was adsorbed intermittently with the PEI. By the third and fourth layer of polyions the polymer islands were essentially merged and protein adsorption as a fourth or fifth layer formed a nearly continuous film. CYP101 was seen to adsorb as globules with a molecular diameter of about 10 nm. CYP2B4 adsorbed to the polyionic films had a slightly elliptical globular shape, also with a molecular diameter of about 10 nm.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Química/instrumentação , Química/métodos , Sistema Enzimático do Citocromo P-450/química , Adsorção , Animais , Ânions , Sítios de Ligação , Cátions , Sistema Enzimático do Citocromo P-450/metabolismo , Citocromos b5/metabolismo , Relação Dose-Resposta a Droga , Íons , Cinética , Microscopia de Força Atômica/métodos , Modelos Moleculares , Poliestirenos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Coelhos , Esteroide Hidroxilases/metabolismoRESUMO
Damage of DNA films after reaction with styrene oxide was detected using derivative square wave voltammetry. Double-stranded (ds) DNA films with initially low backgrounds developed oxidation peaks for DNA bases during incubation with styrene oxide. Films were prepared on pyrolytic graphite (PG) electrodes by casting mixtures of DNA with the poly(ester sulfonic acid) ionomer Eastman AQ38S or by covalent binding of DNA onto oxidized PG. While both types of films gave oxidation peaks in the region 0.6-1.1 V vs SCE after incubations with styrene oxide, DNA/AQ films gave the best signal-to-background ratios. Damage of DNA by reaction with styrene oxide under the electrode incubation conditions was confirmed by capillary electrophoresis. Total integrals of oxidation peaks increased with time of incubation with styrene oxide. Relative peak heights depended on the type of DNA in the order calf thymus ds DNA > salmon sperm ds DNA > supercoiled ds DNA > highly polymerized calf thymus ds DNA.
Assuntos
Dano ao DNA , DNA/química , Animais , Bovinos , Eletroquímica , Compostos de Epóxi/toxicidade , Indicadores e Reagentes , Mutagênicos/toxicidadeRESUMO
This paper describes several applications of polyion-biomolecule films on electrodes related to future development of in vitro chemical toxicity sensors. In the first example, composite films of DNA and ionomers cast onto pyrolytic graphite (PG) electrodes are shown to be useful for detecting DNA damage during incubation with the carcinogen styrene oxide at pH 5.5. Single electrodes can be used to estimate relative damage rates by derivative square wave voltammetry. Films containing the ionomer Nafion gave better reproducibility than another ionomer, Eastman AQ38. In the second example, films containing redox proteins myoglobin (Mb) and cytochrome (cyt) P450cam were constructed in alternate layers with polyions including DNA on rough PG electrodes. Films with reversible protein FeIII/FeII electrochemistry with up to 7 electroactive layers were made. Amounts of electroactive protein on rough PG that were 7 to 17-fold larger than in similar films on smooth gold were achieved because many more layers were electroactive. Films of Mb/DNA also showed oxidation peaks after short incubations with styrene oxide that may be attributable to DNA damage. Results are relevant to the future design of enzyme-DNA films which convert pollutants and drugs to reactive metabolites, followed by electrochemical detection of the resulting DNA damage.
Assuntos
Membranas Artificiais , Toxicologia/métodos , Ânions , Carcinógenos/toxicidade , DNA/análise , Dano ao DNA , PolímerosRESUMO
The chemistry of electrochemically-driven myoglobin-catalyzed oxidation of styrene was investigated in films of DNA or Eastman AQ ionomer on optically transparent electrodes. Conversion of styrene to styrene oxide proceeded via a ferrylmyoglobin radical intermediate. Ferrylmyoglobins were clearly detected by spectroelectrochemistry in films of 1-4 mm thick. The ferrylmyoglobin radical is produced by reaction of metmyoglobin (Mb) in the films with hydrogen peroxide formed by electrochemical catalytic reduction of oxygen catalyzed by Mb. Thus, electrochemically-driven styrene oxidation with these films proceeds by a 'doubly catalytic' electrode-driven reduction-oxidation pathway. Ferrylmyoglobin formation during electrolysis of Mb-DNA films in aerobic solutions was much faster, and styrene oxidation occurred with less Mb decomposition compared to the Mb-AQ films. The better performance of Mb-DNA films is correlated with a larger fraction of electroactive Mb and better stability than for the Mb-AQ films.
Assuntos
DNA/química , DNA/metabolismo , Metamioglobina/química , Metamioglobina/metabolismo , Estireno/química , Estireno/metabolismo , Animais , Bovinos , Eletroquímica , Cavalos , Técnicas In Vitro , Oxirredução , Poliésteres/química , Poliésteres/metabolismo , EspectrofotometriaRESUMO
Direct reversible electron transfer for photosynthetic reaction center from wild type Rhodobacter sphaeroides re-constituted in polycation sandwiched monolayer film was observed in this work. The redox potential E0' = 0.46 V vs. NHE for first primary donor redox couple P/P+ was accurately measured from reversible CV or SWV peaks, which were quite close to those obtained from optic redox titration method. Reaction center (RC) in film was found re-constituted in such an ordered way that the orientation of RC favored the electron transfer in film. Thus, the protein electroactivity seems to be turned on in this artificial biomimic thin film. Furthermore, RC in the film features a photo-induced redox-peak fluctuation, suggesting an intact and functional state for RC in such film. Redox peaks were also found dependent of pH, implying a proton-coupled electron transfer occurring in film. Charge recombination was observed accompanied with change of electrochemical driving force. Electrochemical model assuming several classes of electroactive sites in the films on the electrode with a dispersion of standard potentials successfully fits SWV experimental data at different pulse height and frequency.
Assuntos
Fotossíntese , Complexo de Proteínas do Centro de Reação Fotossintética/química , Rhodobacter sphaeroides/química , Eletroquímica , Eletrodos , Transporte de Elétrons , Concentração de Íons de Hidrogênio , Luz , Membranas Artificiais , Dinâmica não Linear , OxirreduçãoRESUMO
High pressure liquid chromatography (HPLC) using an electrochemical (EC) detector electrode of pyrolytic graphite coated with a film of ionomer Nafion and the water-insoluble surfactant didodecyldimethylammonium bromide (DDAB) was used to achieve separation and detection of all six bromo- and chloro-acetic acids. The Nafion-DDAB film preconcentrates the acid anions facilitating their electrochemical detection by direct reduction at -1.2 V versus SCE. Detection limits were poorer than the EPA-approved GC-ECD method, but HPLC-EC avoids the derivatization necessary for GC. The HPLC-EC method also detected tribromoacetic acid, which has not been determined simultaneously with other halogenated acids by reported chromatographic methods. The HPLC-EC method using a Nafion-DDAB-coated detector electrode gave comparable results to GC-ECD for the determination of TCA in drinking water.
RESUMO
Salt concentration and pH of external solutions were shown to control the electrochemistry of the heme protein myoglobin (MbFe(III)-H2O) in stable, ordered films of didodecyldimethylammonium bromide (DDAB). Protonation of aquometmyoglobin (MbFe(III)-H2O) in these films precedes electron transfer from electrodes, causing formal potentials to shift negative as pH increases from 5 to 8. At pH > 8, MbFe(III)-H2O dissociates to MbFe(III)-OH, which is reduced directly at the electrode at higher rates than MbFe(III)-H2O. Correlations of voltammetric data with FT-IR spectra suggested that at pH < 4.6, an unfolded form of Mb resides in the films and is reduced directly. The concentration of salt in solution influences electrochemical properties of Mb-DDAB films by its influence on Mb conformation and by effects on interfacial Donnan potentials. NMR indicated strong binding of anions to Mb within DDAB films. Bound anions may neutralize positive charge on Mb's surface so that it can reside in a partly hydrophobic environment, as postulated on the basis of previous ESR and linear dichroism studies.
Assuntos
Mioglobina/química , Compostos de Amônio Quaternário/química , Tensoativos/química , Animais , Soluções Tampão , Eletroquímica , Cavalos , Concentração de Íons de Hidrogênio , Cinética , Concentração Osmolar , Conformação Proteica , Sais/química , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Myoglobin (Mb) in thin films of phosphatidyl cholines (PC) or dihexadecyl phosphate (DHP) gave direct, reversible electron transfer between pyrolytic graphite electrodes and the heme Fe(III)/Fe(II) redox couple of the protein. PC films incorporated much more Mb than DHP films. A model assuming several classes of electroactive sites in the films on the electrode with a dispersion of standard potentials successfully fit square-wave voltammetric data at pulse heights > 50 mV. Electron transfer rate constants in PC and DHP films were significantly larger than for Mb in thin films of an insoluble cationic surfactant. The pH dependence of the formal potential of Mb in the PC films suggested that protonation, possibly inducing conformational change, accompanies electron transfer to MbFe(III) between pH 5 and 11. Mb in PC films was used for catalysis of the reduction of trichloroacetic acid.
Assuntos
Membranas Artificiais , Mioglobina/química , Organofosfatos/química , Fosfatidilcolinas/química , Animais , Eletroquímica , Eletrodos , Transporte de Elétrons , Cavalos , Concentração de Íons de Hidrogênio , Cinética , Músculo Esquelético/química , Oxirredução , Tensoativos/química , Ácido Tricloroacético/químicaRESUMO
In previous work, greatly enhanced rates of electron transfer were found for myoglobin (Mb) in ordered films of surfactants on pyrolytic graphite (PG) electrodes. Direct electron transfer is now reported for Mb in films of didodecyldimethylammonium bromide (DDAB) on platinum, tin-doped indium oxide, and gold electrodes. Rates of electron transfer in these films were similar on all electrodes. In the absence of surfactant, electron transfer was observed on bare electrodes only when Mb was purified by chromatography, and only on hydrophilic tin-doped In2O3 or PG. Treatment of tin-doped In2O3 or PG electrodes with unpurified protein solutions blocked electron transfer to Mb in the purified solutions. Reflectance-absorbance infrared and X-ray photoelectron spectroscopy revealed proteinaceous adsorbates on electrodes exposed to unpurified solutions of Mb. This adsorbate blocks electron transfer to Mb and to ferricyanide in solution. Results suggest that electron transfer in the Mb-DDAB films may be facilitated partly by strong adsorption of surfactants on electrodes. Surfactant adsorbed at electrode-film interfaces appears to inhibit adsorption of macromolecules from Mb solutions which could otherwise block electron transfer between Mb and electrodes.
Assuntos
Mioglobina/química , Adsorção , Animais , Eletrodos , Transporte de Elétrons , Cavalos , Substâncias Macromoleculares , TensoativosRESUMO
Oxidation of ferrocene in acetonitrile resulted in films on Pt electrodes under voltammetric conditions. Films were more readily formed with tetrabutylammonium tetrafluoroborate as the electrolyte than with perchlorate salts. No films were detected when ferrocene was oxidized in aqueous 0.05 M cetyltrimethylammonium bromide (CTAB). Analysis of the films by FT-IR and Auger spectroscopy confirmed iron-containing oxidation products on Pt, presumably from chemical reactions of ferricinium ions. Oxidation of 50-100 mM ferrocene in acetonitrile on Pt yielded insoluble precipitates. Analyses by MS, FT-IR, and UV-visible (water extract), suggested a mixture of oligomeric material and a small fraction of ferricinium ions. Film formation had much less influence on voltammograms on 12.5-micron-radius Pt microdisks than on 0.5-mm-radius Pt. This is consistent with the smaller sensitivity of microelectrodes to chemical reactions following charge transfer. The smaller apparent heterogeneous rate constants (k zero') found for ferrocene on macroelectrodes than on microelectrodes could possibly be influenced by film formation on larger Pt electrodes. Correlations between macro- and microelectrode kinetic data suggest that macroelectrode k zero' values may be valid in a relative sense when ohmic drop is negligible. Bias in k zero's on conventional-sized electrodes should be small in solutions giving minimal film formation, such as micellar CTAB.
Assuntos
Compostos Ferrosos/química , Compostos Organometálicos/química , Eletroquímica , Metalocenos , Microeletrodos , Oxirredução , PlatinaRESUMO
Voltammetric reductions of several organohalide pollutants in films of didodecyldimethylammonium bromide (DDAB) and clay-DDAB cast onto pyrolytic graphite electrodes were examined. Direct reduction data show that the amount of accumulation in these liquid crystal films was larger for relatively nonpolar analytes such as trans-1,2-dibromocyclohexane than for chlorinated acids. The vicinal dibromides are probably taken up by hydrophobic regions of the films. Chlorinated acids were accumulated and reduced in DDAB films, but their direct reduction was partly blocked by the clay-DDAB films. Catalytic reductions using films containing metal phthalocyanines had good efficiencies for all substrates studied and shifted reduction potentials positive by 200-550 mV.
RESUMO
Films made from cationic surfactants and well-retained redox catalysts were investigated. Full loading of metal phthalocyaninetetrasulfonates (MPcTS4-) into water-insoluble dialkyldimethylammonium surfactants by ion exchange from aqueous solutions yielded coatings on electrodes that retain these catalyst ions for 1-2 weeks in electrolyte solutions. In contrast, partly loaded films lost most MPcTS4- ions in a few hours. All films showed gel-to-liquid crystal phase transitions at temperatures characteristic of surfactant bilayers. Cross-sectional views by SEM showed layers of 0.1-0.2 micron, as well as some disordered regions. Each larger layer is probably made up of stacks of many molecular bilayers. Retention of MPcTS4- ions seems related to their dimerization. Dimers of MPcTS4- associated with ammonium head groups may crosslink adjacent surfactant bilayers. The MPcTS4- ions that enhance stability in these films are also good redox catalysts.