Rapid Development of New Protein Biosensors Utilizing Peptides Obtained via Phage Display

Abstract

There is a consistent demand for new biosensors for the detection of protein targets, and a systematic method for the rapid development of new sensors is needed. Here we present a platform where short unstructured peptides that bind to a desired target are selected using M13 phage display. The selected peptides are then chemically synthesized and immobilized on gold, allowing for detection of the target using electrochemical techniques such as electrochemical impedance spectroscopy (EIS). A quartz crystal microbalance (QCM) is also used as a diagnostic tool during biosensor development. We demonstrate the utility of this approach by creating a novel peptide-based electrochemical biosensor for the enzyme alanine aminotransferase (ALT), a well-known biomarker of hepatotoxicity. Biopanning of the M13 phage display library over immobilized ALT, led to the rapid identification of a new peptide (ALT5-8) with an amino acid sequence of WHWRNPDFWYLK. Phage particles expressing this peptide exhibited nanomolar affinity for immobilized ALT (Kd,app = 85±20 nM). The newly identified ALT5-8 peptide was then chemically synthesized with a C-terminal cysteine for gold immobilization. The performance of the gold-immobilized peptides was studied with cyclic voltammetry (CV), QCM, and EIS. Using QCM, the sensitivity for ALT detection was 8.9±0.9 Hz/(µg/mL) and the limit of detection (LOD) was 60 ng/mL. Using EIS measurements, the sensitivity was 142±12 impedance percentage change %/(µg/mL) and the LOD was 92 ng/mL. In both cases, the LOD was below the typical concentration of ALT in human blood. Although both QCM and EIS produced similar LODs, EIS is preferable due to a larger linear dynamic range. Using QCM, the immobilized peptide exhibited a nanomolar dissociation constant for ALT (Kd = 20.1±0.6 nM). These results demonstrate a simple and rapid platform for developing and assessing the performance of sensitive, peptide-based biosensors for new protein targets.

An Electrochemical Biosensor Based on a Myoglobin-specific Binding Peptide for Early Diagnosis of Acute Myocardial Infarction

“…18,19 In particular, the combination of electrochemical sensing technology with biomolecule-specific peptides has significantly improved the performance of biosensors for biomarker detection. [20][21][22] Here, we used a short peptide with a sequence of CPSTLGASC (Myo-3R7) as a recognition molecule for capturing myoglobin, a cardiac biomarker. The Myo-3R7 peptide had been screened and identified via phage display.…”

Biomimetic Sensors for Rapid Testing of Water Resources

“…The peptide immobilization resulted in a frequency drop in the QCM signal due to the increased mass loading, a drop in current was detectable by CV, and an impedance increase in the EIS due to the blocking of the gold surface. The packing density of the surface-bound peptide can be calculated using the Sauerbrey equation [13]:…”

“…The quantitative response achieved for both QCM and EIS validates that both methods could be used as a sensing technique in quantitative ALT measurements. In addition to the generation of this ALT-specific recognition element, we have developed a recognition element for the cardiac specific biomarker, troponin I (TnI) [13,14]. Again, phage display was employed to generate the recognition peptide while QCM and EIS validated and quantified the binding events and specificity of the peptide for the target, TnI.…”

Selection of High Affinity Peptides for Prediction of Colorectal Adenoma-to-Carcinoma Progression

“…Thus, we have chosen LRG1 and TTR as target proteins to identify novel peptide motifs via phage display technique. Evolutionary phage display is a powerful method which enables the isolation of novel peptides for the desired targets . Compared to antibody‐based immunoassay, linear and unstructured short peptides have attracted to be effective material for developing new biosensor in clinical applications.…”