High affinity peptides for the recognition of the heart disease biomarker troponin I identified using phage display

Abstract

Troponin I is a specific and sensitive clinical biomarker for myocardial injury. In this study we have used polyvalent phage display to isolate unique linear peptide motifs which recognize both the human and rat homologs of troponin I. The peptide specific for human troponin I has a sequence of FYSHSFHENWPS and the peptide specific for the rat troponin I has a sequence of FHSSWPVNGSTI. Enzyme-linked immunosorbent assays (ELISAs) were used to evaluate the binding interactions, and the two phage-displayed peptides exhibited some cross-reactivity, but they were both more specific for the troponin I homolog they were selected against. The binding affinities of the phage-displayed peptides were decreased by the presence of complex tissue culture media (MEM), and the addition of 10% calf serum further interfered with the binding of the target proteins. Kinetic indirect phage ELISAs revealed that both troponin I binding peptides were found to have nanomolar affinities for the troponin proteins while attached to the phage particles. To our knowledge, this is the first example of isolation and characterization of troponin I binders using phage display technology. These new peptides may have potential utility in the development of new clinical assays for cardiac injury as well as in monitoring of cardiac cells grown in culture.

Highly Sensitive Electrochemiluminescence Assay for Cardiac Troponin I and Adenosine Triphosphate by using Supersandwich Amplification and Bifunctional Aptamer

“…BSA was purchased from J&K Scientific Ltd. Myoglobin(myo), 6-mercapto-1-hexanol (MCH), Ru(phen) 3 Cl 2 , Bis(2,2'-bipyridine)-4'-methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester-bis(hexafluorophosphate) (Ru1), adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), thymidine triphosphate (TTP), tripropylamine (TPA) and glycerol were obtained from Sigma-Aldrich. Boric acid, alcohol, formaldehyde, ammonium persulfate, tetramethylethylene-diamine, glacial acetic acid and acrylamide were obtained from Sinopharm Chemical Reagent Co., Ltd. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Specific peptide of cTnI (CFYSHSFHENWPSK, MW = 1768.91, Figure S-8 in Supporting Information) for cTnI, designed according to References, [27,35] was obtained from Shanghai Apeptide Co., Ltd. Oligonucleotides were synthesized by Takara Biotechnology Co. Ltd. and the sequences were following: The pre-hybridized S2-S3 solution (10 mM) was prepared by mixing 10 mL of 100 mM S2 and 10 mL of 100 mM S3 in 80 mL of DNA hybridization buffer, then heating the solution to 90 8C, and allowing it to cool slowly to room temperature. Ru1 labelled S4 was synthesized according to reference.…”

“…The specific peptide of cTnI was designed according to Park et al, which can selectively bind to cTnI with a disassociation constant at nanomolar level. [27] A sandwich-type conjugate (peptide < cTnI > S1) was formed when the peptide modified electrode was successively reacted with target cTnI and S1 probe. Then, the hybridizations between S1 and two ss-DNA auxiliary probes (S2 and S3) lead to the formation of longrange ds-DNA polymers on the surface of the electrode.…”

Biomimetic Sensors for Rapid Testing of Water Resources

“…In an effort to use an electrode material both more resistant to biofouling and possessing a larger potential range compared to noble metal electrodes [15][16][17], we developed a new method for the fabrication of carbon paste electrodes (CPEs) that optimizes their stability in a flow-based microfluidic device [18]. The greater stability of these CPEs results from exploiting beneficial properties of both soft and rigid composites, which are used as the binder, during CPE fabrication.…”

Sensitive electrogenerated chemiluminescence peptide-based biosensor for the determination of troponin I with gold nanoparticles amplification

“…Short linear binding peptides, which are obtained using phage display, have received considerable interest in protein analysis due to the advantages, such as stable, resistant to harsh environments, and more amenable to engineering at the molecular level than antibodies [16]. Recently, Park et al reported a new peptide (FYSHSFHENWPS) that selectively bound to TnI with a disassociation constant of the complex in nanomolar level [17]. We developed two homogeneous ECL peptide-based methods for the determination of TnI using this peptide as a molecular recognition element [14,15].…”

“…Two specific peptides including peptide1 with a sequence of CFYSHSFHENWPS, in which a thiol-containing cysteine residue was incorporated at the end of the specific peptide to facilitate self-assembly onto the surface of gold, peptide2 with a sequence of FYSHSFHENWPSK, in which a NH 2 -containing lysine residue was incorporated at the end of the peptide to covalently couple with ECL signal, were designed according to ref. [17] and employed as capture peptide and report peptide, respectively. The peptide2 was labeled with ruthenium bis(2,2′-bipyridine) (2,2′-bipyridine-4,4′-dicarboxylic acid)-N -hydroxysuccinimide ester (Ru(bpy) 2 (dcbpy)NHS) via acylation reaction through NH 2 -containing lysine at the end of the peptide to form the ECL probe Ru-peptide2.…”