RESUMEN
The sensitive detection of cancer biomarkers in urine could revolutionize cancer diagnosis and treatment. Such detectors must be inexpensive, easy to interpret, and sensitive. This report describes a bioaffinity matrix of viruses integrated into PEDOT films for electrochemical sensing of prostate-specific membrane antigen (PSMA), a prostate cancer biomarker. High sensitivity to PSMA resulted from synergistic action by two different ligands to PSMA on the same phage particle. One ligand was genetically encoded, and the secondary recognition ligand was chemically synthesized to wrap around the phage. The dual ligands result in a bidentate binder with high-copy, dense ligand display for enhanced PSMA detection through a chelate-based avidity effect. Biosensing with virus-PEDOT films provides a 100 pM limit of detection for PSMA in synthetic urine without requiring enzymatic or other amplification.
Asunto(s)
Alquinos , Antígenos de Superficie/orina , Azidas , Biomarcadores de Tumor/orina , Técnicas Biosensibles/métodos , Glutamato Carboxipeptidasa II/orina , Neoplasias de la Próstata/orina , Compuestos de Sulfhidrilo , Alquinos/química , Azidas/química , Bacteriófagos/química , Bacteriófagos/genética , Ciclización , Humanos , Ligandos , Límite de Detección , Masculino , Modelos Moleculares , Estructura Molecular , Compuestos de Sulfhidrilo/químicaRESUMEN
We demonstrate the de novo fabrication of a biosensor, based upon virus-containing poly(3,4-ethylene-dioxythiophene) (PEDOT) nanowires, that detects prostate-specific membrane antigen (PSMA). This development process occurs in three phases: (1) isolation of a M13 virus with a displayed polypeptide receptor, from a library of ≈10(11) phage-displayed peptides, which binds PSMA with high affinity and selectivity, (2) microfabrication of PEDOT nanowires that entrain these virus particles using the lithographically patterned nanowire electrodeposition (LPNE) method, and (3) electrical detection of the PSMA in high ionic strength (150 mM salt) media, including synthetic urine, using an array of virus-PEDOT nanowires with the electrical resistance of these nanowires for transduction. The electrical resistance of an array of these nanowires increases linearly with the PSMA concentration from 20 to 120 nM in high ionic strength phosphate-buffered fluoride (PBF) buffer, yielding a limit of detection (LOD) for PSMA of 56 nM.
Asunto(s)
Antígenos de Superficie/análisis , Antígenos de Superficie/inmunología , Bacteriófago M13/inmunología , Técnicas Biosensibles/instrumentación , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Glutamato Carboxipeptidasa II/análisis , Glutamato Carboxipeptidasa II/inmunología , Nanocables/química , Polímeros/química , Neoplasias de la Próstata/diagnóstico , Secuencia de Aminoácidos , Bacteriófago M13/aislamiento & purificación , Diseño de Equipo , Humanos , Límite de Detección , Masculino , Biblioteca de Péptidos , Péptidos/química , Péptidos/inmunología , Neoplasias de la Próstata/inmunologíaRESUMEN
Virus-poly(3,4-ethylenedioxythiophene) (virus-PEDOT) biocomposite films are prepared by electropolymerizing 3,4-ethylenedioxythiophene (EDOT) in aqueous electrolytes containing 12 mM LiClO(4) and the bacteriophage M13. The concentration of virus in these solutions, [virus](soln), is varied from 3 to 15 nM. A quartz crystal microbalance is used to directly measure the total mass of the biocomposite film during its electrodeposition. In combination with a measurement of the electrodeposition charge, the mass of the virus incorporated into the film is calculated. These data show that the concentration of the M13 within the electropolymerized film, [virus](film), increases linearly with [virus](soln). The incorporation of virus particles into the PEDOT film from solution is efficient, resulting in a concentration ratio of [virus](film):[virus](soln) ≈ 450. Virus incorporation into the PEDOT causes roughening of the film topography that is observed using scanning electron microscopy and atomic force microscopy (AFM). The electrical conductivity of the virus-PEDOT film, measured perpendicular to the plane of the film using conductive tip AFM, decreases linearly with virus loading, from 270 µS/cm for pure PEDOT films to 50 µS/cm for films containing 100 µM virus. The presence on the virus surface of displayed affinity peptides did not significantly influence the efficiency of incorporation into virus-PEDOT biocomposite films.
Asunto(s)
Bacteriófago M13/química , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Polímeros/química , Electroquímica , Galvanoplastia , Compuestos de Litio/química , Polimerizacion , Propiedades de SuperficieRESUMEN
Composite films composed of poly(3,4-ethylenedioxythiophene), PEDOT, and the filamentous virus M13-K07 were prepared by electrooxidation of 3,4-ethylenedioxythiophene (EDOT) in aqueous solutions containing 8 nM of the virus at planar gold electrodes. These films were characterized using atomic force microscopy and scanning electron microscopy. The electrochemical impedance of virus-PEDOT films increases upon exposure to an antibody (p-Ab) that selectively binds to the M13 coat peptide. Exposure to p-Ab causes a shift in both real (Z(RE)) and imaginary (Z(IM)) impedance components across a broad range of frequencies from 50 Hz to 10 kHz. Within a narrower frequency range from 250 Hz to 5 kHz, the increase of the total impedance (Z(total)) with p-Ab concentration conforms to a Langmuir adsorption isotherm over the concentration range from from 6 to 66 nM, yielding a value for K(d) = 16.9 nM at 1000 Hz.
Asunto(s)
Bacteriófago M13/química , Técnicas Biosensibles/métodos , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Polímeros/química , Anticuerpos/análisis , Anticuerpos/inmunología , Bacteriófago M13/inmunología , Impedancia Eléctrica , Galvanoplastia , Oro/química , Límite de DetecciónRESUMEN
The separate fields of conducting polymer-based electrochemical sensors and virus-based molecular recognition offer numerous advantages for biosensing. Grafting M13 bacteriophage into an array of poly (3,4-ethylenedioxythiophene) (PEDOT) nanowires generated hybrids of conducting polymers and viruses. The virus incorporation into the polymeric backbone of PEDOT occurs during electropolymerization via lithographically patterned nanowire electrodeposition. The resultant arrays of virus-PEDOT nanowires enable real-time, reagent-free electrochemical biosensing of analytes in physiologically relevant buffers.
Asunto(s)
Técnicas Biosensibles , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Nanocables , Polímeros/química , Bacteriófago M13/química , Microscopía de Fuerza Atómica , Microscopía Electrónica de Rastreo , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
M13 phage have provided scaffolds for nanostructure synthesis based upon self-assembled inorganic and hard materials interacting with phage-displayed peptides. Additionally, phage display has been used to identify binders to plastic, TiO(2), and other surfaces. However, synthesis of phage-based materials through the hybridization of soft materials with the phage surface remains unexplored. Here, we present an efficient "phage wrapping" strategy for the facile synthesis of phage coated with soluble, cationic polymers. Polymers bearing high positive charge densities demonstrated the most effective phage wrapping, as shown by assays for blocking nonspecific binding of the anionic phage coat to a high pI target protein. The results establish the functional group requirements for hybridizing phage with soft materials and solve a major problem in phage display-nonspecific binding by the phage to high pI target proteins.