RESUMO
The carbohydrate antigen 19-9 (CA 19-9) is the most commonly used biomarker in the clinical diagnosis of pancreatic cancer. Multiphoton nonlinear laser wave-mixing spectroscopy is presented as an ultrasensitive detection method for CA 19-9. Wave mixing is an optical absorption-based method, and hence, one can detect CA 19-9 without labels in their native form using compact ultraviolet (UV) lasers or labeled samples using a visible laser. The wave-mixing signal exhibits a quadratic dependence on the sample concentration, and hence, it is an ideal sensor to monitor small changes in the sample. Wave mixing has inherent advantages over other absorption-based detection methods, including short optical path length (micrometer-thin samples instead of 1 cm cuvette) and excellent spatial resolution (micrometer probe). Since the laser wave-mixing probe volume is small (picoliter), it is convenient to interface to microfluidics or capillary-based electrophoresis systems to enhance chemical specificity. Our wave-mixing detectors could be configured as portable battery-powered devices suitable for field use. Laser wave-mixing spectroscopy offers enhanced selectivity levels for protein detection when coupled with capillary electrophoresis (CE). We report a concentration detection limit of 0.16 U/mL, and a corresponding mass detection limit of 1.2 × 10-8 U, and these detection limits are better than those of chemiluminescence- or ELISA- based methods.
RESUMO
Nonlinear laser wave-mixing spectroscopy is demonstrated as a fast and sensitive detection method for heart-failure biomarkers, pro-atrial natriuretic peptide (proANP) and brain natriuretic peptide (BNP). Wave mixing is an ultrasensitive optical absorption-based method and analytes can be detected in their native form or labeled with fluorophore and chromophore labels. In this study, we utilized Chromeo P540 dye to label the peptides for wave-mixing detection. The wave-mixing signal is created from the diffraction of incoming photons by the thermal grating at the capillary analyte cell. The signal beam is strong, collimated, and coherent (laser-like) and it is collected using a simple photodetector with an excellent signal-to-noise ratio. We demonstrated advantages of this technique over conventional assays including shorter analysis times, smaller sample requirements, and higher throughput. To enhance detection selectivity and sensitivity levels, wave mixing is effectively coupled to capillary zone electrophoresis (CZE) and field-amplified sample stacking (FASS) methods. We determined detection limits of 7.4 × 10-10 M or 55 zmol and 6.8 × 10-10 M or 51 zmol for proANP and BNP, respectively, and separated and detected both peptides within 2 min. Due to the challenges in the confirmatory diagnoses of heart failure, wave-mixing serves as a potentially beneficial screening tool in addition to the commonly used echocardiographic tests.
Assuntos
Insuficiência Cardíaca , Peptídeos Natriuréticos , Humanos , Insuficiência Cardíaca/diagnóstico , Análise Espectral , Lasers , PeptídeosRESUMO
It is well documented that blue light absorption by bacterial chromophores triggers downstream production of reactive oxygen species (ROS), which in turn results in bacterial cell death. To elucidate the importance of chromophores in the bactericidal effect of blue light, and to determine whether blue light absorption per se or the presence of porphyrins known to engender ROS is crucial in blue light treatment, we studied the effect of 450 nm pulsed light on Streptococcus agalactiae, also known as Group B Streptococcus (GBS) strain COH1. GBS does not synthesize porphyrins but has a blue light-absorbing chromophore, granadaene. We irradiated planktonic cultures of GBS with or without exogenous chromophore supplementation using either protoporphyrin IX (PPIX), coproporphyrin III (CPIII), Nicotinamide adenine dinucleotide (NAD), reduced nicotinamide adenine dinucleotide (NADH), Flavin adenine dinucleotide (FAD), or Flavin mononucleotide (FMN). Quantification of surviving bacterial colonies, presented as percent survival and CFU/mL (log10), showed that (1) 450 nm blue light does not suppress the growth of GBS, even though its endogenous chromophore, granadaene, absorbs light in the 450 nm spectrum. (2) The addition of either of the two exogenous porphyrins, PPIX or CPIII, significantly suppressed GBS, indicating the importance of porphyrins in the antimicrobial action of blue light. (3) Adding exogenous FMN or FAD, two known absorbers of 450 nm light, minimally potentiated the bactericidal effect of blue light, again confirming that mere absorption of blue light by chromophores does not necessarily result in bacterial suppression. (4) Irradiation of GBS with or without NAD+ or NADH supplementation-two weak absorbers of 450 nm light-minimally suppressed GBS, indicating that a blue light-absorbing chromophore is essential for the bactericidal action of blue light. (5) Collectively, these findings show that in addition to the presence of a blue light-absorbing chromophore in bacteria, a chromophore with the right metabolic machinery and biochemical structure, capable of producing ROS, is necessary for 450 nm blue light to suppress GBS.
Assuntos
Luz , Porfirinas/química , Porfirinas/farmacologia , Streptococcus agalactiae/efeitos dos fármacos , Streptococcus agalactiae/efeitos da radiação , Interações Medicamentosas , Flavina-Adenina Dinucleotídeo/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/efeitos da radiação , NAD/farmacologia , Streptococcus agalactiae/fisiologiaRESUMO
An ultrasensitive label-free antibody-free detection method for malachite green and crystal violet is presented using nonlinear laser wave-mixing spectroscopy and capillary zone electrophoresis. Wave-mixing spectroscopy provides a sensitive absorption-based detection method for trace analytes. This is accomplished by forming dynamic gratings within a sample cell, which diffracts light to create a coherent laser-like signal beam with high optical efficiency and high signal-to-noise ratio. A cubic dependence on laser power and square dependence on analyte concentration make wave mixing sensitive enough to detect molecules in their native form without the use of fluorescent labels for signal enhancement. A 532 nm laser and a 635 nm laser were used for malachite green and crystal violet sample excitation. The use of two lasers of different wavelengths allows the method to simultaneously detect both analytes. Selectivity is obtained through the capillary zone electrophoresis separation, which results in characteristic migration times. Measurement in capillary zone electrophoresis resulted in a limit of detection of 6.9 × 10(-10)M (2.5 × 10(-19) mol) for crystal violet and 8.3 × 10(-11)M (3.0 × 10(-20) mol) for malachite green at S/N of 2.
Assuntos
Eletroforese Capilar/métodos , Violeta Genciana/análise , Corantes de Rosanilina/análise , Violeta Genciana/química , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes , Corantes de Rosanilina/químicaRESUMO
Multi-photon nonlinear laser wave-mixing spectroscopy is a novel absorption-based technique that offers excellent detection sensitivity for biomedical applications, including early diagnosis and investigation of neurodegenerative diseases. α-Synuclein is linked to Parkinson's disease (PD), and characterization of its oligomers and quantification of the protein may contribute to understanding PD. The laser wave-mixing signal has a quadratic dependence on analyte concentration, and hence the technique is effective in monitoring small changes in concentration within biofluids. A wide variety of labels can be employed for laser wave-mixing detection due to its ability to detect both chromophores and fluorophores. In this investigation, two fluorophores and a chromophore are studied and used as labels for the detection of α-synuclein. Wave-mixing detection limits of PD-related protein conjugated with fluorescein isothiocyanate, QSY 35 acetic acid, succinimidyl ester, and Chromeo P503 were determined to be 1.4 × 10(-13) M, 1.4 × 10(-10) M, and 1.9 × 10(-13) M, respectively. Based on the laser probe volume used, the corresponding mass detection limits were determined to be 1.1 × 10(-23) mol, 1.1 × 10(-20) mol, and 1.5 × 10(-23) mol. This study also presents molecular-based separation and quantification of α-synuclein by laser wave mixing coupled with capillary electrophoresis.
Assuntos
Eletroforese Capilar/métodos , alfa-Sinucleína/análise , Sequência de Aminoácidos , Peso Molecular , Dinâmica não Linear , Espectrofotometria Ultravioleta , alfa-Sinucleína/químicaRESUMO
Sensitive absorption-based detection of anthracycline antibiotics, daunorubicin and doxorubicin is demonstrated using a capillary electrophoresis system interfaced to a nonlinear wave-mixing detection system. Unlike conventional absorption methods, this nonlinear absorption method can detect very thin analytes (50 microm) efficiently. At the same peak height, the wave-mixing CE peak is narrower than a conventional CE peak, and hence, compared to other laser-based or non-laser-based CE on-column detection methods, our wave-mixing detection method offers intrinsically enhanced separation resolution even when using identical CE separation conditions. In this unusually sensitive "absorbance" detection method, two input laser beams interact to produce a thermally induced grating from which coherent laser-like wave-mixing signal beams are created. Using our sensitive "absorbance" on-column CE detector, we report a preliminary concentration detection limit of 9.9x10(-10)M using a 50-mum i.d. capillary column. The corresponding "injected" mass detection limit is 9.1x10(-18)mol using an injection volume of 9.2nL. The corresponding preliminary "detected" mass detection limit inside the 12-pL detector probe volume is 1.2x10(-20)M.
Assuntos
Antraciclinas/análise , Eletroforese Capilar/métodos , Preparações Farmacêuticas/química , Absorção , Antibióticos Antineoplásicos/análise , Daunorrubicina/análise , Doxorrubicina/análise , Eletroforese Capilar/instrumentação , Lasers , Limite de DetecçãoRESUMO
A circular dichroism (CD) detector based on laser four-wave mixing (FWM) is demonstrated using separate injections of analyte enantiomers onto a standard silica-based microbore high-performance liquid chromatography (HPLC) column. Using the chiral column, a preliminary "detected" mass detection limit of 180 pg is determined inside a laser probe volume of 200 pL, corresponding to a circular dichroism detection limit, DeltaA, of 2.2 x 10(-5) for (-) camphorquinone. Detection sensitivity levels are dramatically improved when our FWM-CD detector is interfaced to a microbore system due to the lower mobile-phase flow rates and the smaller sample concentrations required for the analysis. Using the microbore column, a preliminary circular dichroism detection limit, DeltaA, of 1.6 x 10(-6) and a preliminary concentration detection limit of 4.1 x 10(-4) M are determined for camphorquinone. This corresponds to a "detected" mass detection limit of 33 pg for the chiral compound. Laser wave mixing offers better detection limits than conventional circular dichroism detection methods and, hence, offers many promising applications.
Assuntos
Cromatografia Líquida de Alta Pressão/instrumentação , Dicroísmo Circular/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Dicroísmo Circular/métodos , Desenho de Equipamento , Peso Molecular , Sensibilidade e Especificidade , Estereoisomerismo , Terpenos/análise , Terpenos/químicaRESUMO
Nonlinear laser wave mixing is presented as an ultrasensitive absorption-based method for the detection of proteins and antibodies using a nonfluorescing chromophore label, Coomassie Brilliant Blue (CBB). The complexes are flowed through a 150-microm (i.d.) capillary cell and detected using a low-power He-Ne laser. The wave-mixing signal is detected after 10 min of room temperature incubation for the antibody complex and after 18 min for the protein complex. All solutions are prepared in an aqueous buffer without the addition of organic modifiers. Concentration detection limits of 3.4 x 10(-19) and 6.4 x 10(-14) M (signal-to-noise ratio [S/N] = 2) are determined for bovine serum albumin (BSA) and human papillomavirus (HPV) antibody, respectively. Based on the small laser probe volume used (i.e., overlap volume of the two input beams), mass detection limits of 1.7 x 10(-22) and 2.6 x 10(-17) mol are determined for BSA and HPV antibody, respectively.
Assuntos
Anticorpos/análise , Lasers de Gás , Proteínas/análise , Corantes de Rosanilina/química , Animais , Anticorpos/química , Bovinos , Humanos , Indicadores e Reagentes , Papillomaviridae/imunologia , Ligação Proteica , Soroalbumina Bovina/análise , Soroalbumina Bovina/química , Espectrofotometria UltravioletaRESUMO
Nonlinear laser wave mixing in a common graphite furnace atomizer is presented as a zeptomole-level, sub-Doppler, high-resolution atomic absorption spectrophotometric method. A nonplanar three-dimensional wave-mixing optical setup is used to generate the signal beam in its own space. Signal collection is efficient and convenient using a template-based optical alignment. The graphite furnace atomizer offers advantages including fast and convenient introduction of solid, liquid, or gas analytes, clean atomization environment, and minimum background noise. Taking advantage of the unique features of the wave-mixing optical method and those of the graphite furnace atomizer, one can obtain both excellent spectral resolution and detection sensitivity. A preliminary concentration detection limit of 0.07 parts-per-quadrillion and a preliminary mass detection limit of 0.7 ag or 8 zmol are determined for rubidium using a compact laser diode as the excitation source.
Assuntos
Lasers , Espectrofotometria Atômica/métodos , Grafite , Matemática , Rubídio/análise , Rubídio/química , Sensibilidade e Especificidade , Espectrofotometria Atômica/instrumentação , Temperatura , Fatores de TempoRESUMO
Nonlinear laser wave mixing is a versatile spectroscopic method for trace elemental analysis at high spectral resolution. Sub-Doppler spectral resolution allows isotope and hyperfine structure measurements of some of the elements even when using a room-pressure analytical flame (i.e., sub-Doppler but Lorentzian broadened spectra). A non-planar wave-mixing optical setup offers some advantages as compared to the conventional planar wave-mixing setup including better signal collection efficiency and easier optical alignment. Using our absorption-based wave mixing, a detection limit of 0.05 ng/mL (i.e., 50 parts-per-trillion) is reported for Rb in an air/acetylene flame, while still maintaining sub-Doppler spectral resolution for the infrared 780.0-nm Rb transition line.