Label-free detection of protein-protein interactions using a calmodulin-modified nanowire transistor

Abstract

In this study, we describe a highly sensitive and reusable silicon nanowire field-effect transistor for the detection of protein-protein interactions. This reusable device was made possible by the reversible association of glutathione S-transferase-tagged calmodulin with a glutathione modified transistor. The calmodulin-modified transistor exhibited selective electrical responses to Ca 2þ (≥1 μM) and purified cardiac troponin I (∼7 nM); the change in conductivity displayed a linear dependence on the concentration of troponin I in a range from 10 nM to 1 μM. These results are consistent with the previously reported concentration range in which the dissociation constant for the troponin I-calmodulin complex was determined. The minimum concentration of Ca 2þ required to activate calmodulin was determined to be 1 μM. We have also successfully demonstrated that the N-type Ca 2þ channels, expressed by cultured 293T cells, can be recognized specifically by the calmodulin-modified nanowire transistor. This sensitive nanowire transistor can serve as a high-throughput biosensor and can also substitute for immunoprecipitation methods used in the identification of interacting proteins.calcium ion | glutathione S-transfrease | N-type calcium channel | silicon nanowire field-effect transistor

Preparation of Silicon Nanowire Field-effect Transistor for Chemical and Biosensing Applications

“…As shown in Table 1, several studies have used NWFET-based biosensors for label-free, ultra-high-sensitivity, and real-time detection of various biological targets, including a single virus 2 , adenosine triphosphate and kinase binding 3 , neuronal signals 4 , metal ions 5,6 , bacterial toxins 7 , dopamine 8 , DNA [9][10][11] , RNA 12,13 , enzyme and cancer biomarkers [14][15][16][17][18][19] , human hormones 20 , and cytokines 21,22 . These studies have demonstrated that NWFET-based biosensors represent a powerful detection platform for a broad range of biological and chemical species in a solution.…”

Recent Advances in Calcium/Calmodulin-Mediated Signaling with an Emphasis on Plant-Microbe Interactions

“…1). The reusable device is made possible by the reversible association of glutathione S-transferase-tagged CaM with a glutathione-modified transistor (Lin et al, 2010). The minimum concentration of Ca 2+ required to activate CaM is 1 mM, and this sensitive nanowire transistor can serve as a high-throughput biosensor and substitute for immunoprecipitation methods used in the identification of interacting proteins.…”

“…These target proteins are involved in almost all aspects of plant growth and development as well as in responses to abiotic and biotic stresses. Recently, an mRNA display technique and a CaM-modified nanowire transistor method have been developed for isolating CaM-binding proteins in humans (Shen et al, 2005;Lin et al, 2010). The mRNA display technique to identify Ca 2+ /CaM-binding proteins is to screen the mRNA-displayed proteome libraries with biotinylated CaM.…”

Specific detection of biomolecules in physiological solutions using graphene transistor biosensors

“…In particular, field-effect transistor (FET) biosensors configured from semiconducting nanowires (1, 2), single-walled carbon nanotubes (1, 3, 4), and graphene (1, 5, 6) have been extensively investigated since the first report of real-time protein detection using silicon nanowire devices (7). Subsequent studies have demonstrated highly sensitive and in some cases multiplexed detection of key analytes, including protein disease markers (8-10), nucleic acids (11-13), and viruses (14), as well as detection of protein-protein interactions (8,(15)(16)(17) and enzymatic activity (8).The success achieved with nanomaterial-based FET biosensors has been limited primarily to measurements in relatively low-ionicstrength nonphysiological solutions due to the Debye screening length (18,19). In short, the screening length in physiological solutions, <1 nm, reduces the field produced by charged macromolecules at the FET surface and thus makes real-time label-free detection difficult.…”

“…In particular, field-effect transistor (FET) biosensors configured from semiconducting nanowires (1, 2), single-walled carbon nanotubes (1, 3, 4), and graphene (1, 5, 6) have been extensively investigated since the first report of real-time protein detection using silicon nanowire devices (7). Subsequent studies have demonstrated highly sensitive and in some cases multiplexed detection of key analytes, including protein disease markers (8-10), nucleic acids (11-13), and viruses (14), as well as detection of protein-protein interactions (8,(15)(16)(17) and enzymatic activity (8).…”