RESUMEN
We developed and validated a reliable, robust, and easy-to-implement quantitative method for multielemental analysis of low-volume samples. Our ICP-MS-based method comprises the analysis of 20 elements (Mg, P, S, K, Ca, V, Cr, Mn, Fe, Co, Cu, Zn, Se, Br, Rb, Sr, Mo, I, Cs, and Ba) in 10 µL of serum and 12 elements (Mg, S, Mn, Fe, Co, Cu, Zn Se, Br, Rb, Mo, and Cs) in less than 250â¯000 cells. As a proof-of-concept, we analyzed the elemental profiles of serum and sorted immune T cells derived from naiÌve and tumor-bearing mice. The results indicate a tumor systemic effect on the elemental profiles of both serum and T cells. Our approach highlights promising applications of multielemental analysis in precious samples such as rare cell populations or limited volumes of biofluids that could provide a deeper understanding of the essential role of elements as cofactors in biological and pathological processes.
Asunto(s)
Compuestos Inorgánicos/análisis , Espectrometría de Masas/métodos , Neoplasias/química , Animales , Línea Celular Tumoral , Cobre/análisis , Cobre/sangre , Compuestos Inorgánicos/sangre , Límite de Detección , Magnesio/análisis , Magnesio/sangre , Ratones , Ratones Endogámicos C57BL , Neoplasias/patología , Linfocitos T/química , Linfocitos T/citología , Linfocitos T/metabolismo , Trasplante Homólogo , Zinc/análisis , Zinc/sangreRESUMEN
Both protein and solid-state nanopores are under intense investigation for the analysis of nucleic acids. A crucial advantage of protein nanopores is that site-directed mutagenesis permits precise tuning of their properties. Here, by augmenting the internal positive charge within the alpha-hemolysin pore and varying its distribution, we increase the frequency of translocation of a 92-nt single-stranded DNA through the pore at +120 mV by approximately 10-fold over the wild-type protein and dramatically lower the voltage threshold at which translocation occurs, e.g., by 50 mV for 1 event.s(-1).muM(-1). Further, events in which DNA enters the pore, but is not immediately translocated, are almost eliminated. These experiments provide a basis for improved nucleic acid analysis with protein nanopores, which might be translated to solid-state nanopores by using chemical surface modification.
Asunto(s)
Toxinas Bacterianas/química , ADN de Cadena Simple/química , Proteínas Hemolisinas/química , Nanoestructuras/química , Nanotecnología , Toxinas Bacterianas/genética , Transporte Biológico , Ingeniería Genética , Proteínas Hemolisinas/genética , Mutación Puntual , Porosidad , Pliegue de Proteína , Propiedades de SuperficieRESUMEN
Detection of chemical processes on a single molecule scale is the ultimate goal of sensitive analytical assays. We recently reported the possibility to detect chemical modifications on individual molecules by monitoring a change in the single ion channel conductance of derivatives of gramicidin A (gA) upon reaction with analytes in solution. These peptide-based nanosensors detect reaction-induced changes in the charge of gA derivatives that were engineered to carry specific functional groups near their C-terminus.1 Here, we discuss five key design parameters to optimize the performance of such chemomodulated ion channel sensors. In order to realize an effective sensor that measures changes in charge of groups attached to the C-terminus of a gA pore, the following conditions should be fulfilled: (1) the change in charge should occur as close to the entrance of the pore as possible; (2) the charge before and after reaction should be well-defined within the operational pH range; (3) the ionic strength of the recording buffer should be as low as possible while maintaining a detectable flow of ions through the pore; (4) the applied transmembrane voltage should be as high as possible while maintaining a stable membrane; (5) the lipids in the supporting membrane should either be zwitterionic or charged differently than the derivative of gA. We show that under the condition of high applied transmembrane potential (>100 mV) and low ionic strength of the recording buffer (< or =0.10 M), a change in charge at the entrance of the pore is the dominant requirement to distinguish between two differently charged derivatives of gA; the conductance of the heterodimeric gA pore reported here does not depend on a difference in charge at the exit of the pore. We provide a simple explanation for this asymmetric characteristic based on charge-induced local changes in the concentration of cations near the lipid bilayer membrane. Charge-based ion channel sensors offer tremendous potential for ultrasensitive functional detection since a single chemical modification of each individual sensing element can lead to readily detectable changes in channel conductance.