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 (K(d,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 (K(d) = 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.

Figure 1. Schematic diagram illustrating the general process of biosensor development.

(A) Work flow diagram for biosensor development: 1. Target protein selection, 2. Phage display selection, 3. Peptide synthesis, 4. QCM diagnosis, 5. Biosensor detection. B) The basic principle of QCM where the binding of the target protein to the immobilized peptides causes a frequency change in the oscillation of the quartz crystal. C) The basic principle of EIS where the binding of the target protein to the immobilized peptides causes increased resistance to the reaction of an added redox couple. See text for details.

Figure 2. Characterization of the selected ALT binding phage clones using ELISA.

ALT binding of the selected phage clones ALT5-8 (▪), ALT4-3 (□), ALT5-9 (▴) and M13 control phage (▵) with varying concentrations of phage particles ranging from 10⁵ pfu to 10¹¹ pfu. All measurements were performed in triplicate and error bars represent standard deviations.

Figure 3. Determination of the apparent dissociation constant of ALT5-8 for ALT.

ELISA experiments were performed in triplicate at eight different phage concentrations and the data were fit by a Langmuir isotherm (R² = 0.88) in order to estimate the apparent equilibrium dissociation constant (K_d,app = 85±20 nM.).

Figure 4. Lineweaver-Burk plots for the determination the of the ALT enzyme inhibition by the ALT5-8 peptide.

Varying concentrations of peptide [I] were added as a function of initial ALT concentration [E]. All data were collected in triplicate and R² values are shown for the best fit line for each inhibitor concentration. A K_I value of 71±17 nM was obtained by fitting the entire data set simultaneously to the bi-bi ping pong rate equation with competitive inhibition (Eqn. 2).

Figure 5. Sensor preparation.

A) the change in frequency during the immobilization of peptide (Pep, 0.1 mM), rinsing (Rinse) and subsequent blocking with cysteine (Cys, 1 mM) on gold using QCM; B) CV of the bare gold electrode (Au), the electrode with the peptide (Au-Pep), and the blocked electrode (Au-Pep-Cys), C) EIS of bare gold (Au), the peptide modified electrode (Au-Pep) and the blocked electrode (Au-Pep-Cys) in a 1 mM solution of Fe(CN)₆ ^4-/3- in 0.1 M NaClO₄.

Figure 6. Sensor operation.

A) QCM frequency change of Au-Pep-Cys electrode in Buffer alone and in 10 µg/mL ALT, B) CV of Au-Pep-Cys electrode before (Baseline) and after Buffer alone or ALT binding, C) EIS of Au-Pep-Cys before (Baseline) and after Buffer alone or ALT binding. After incubation in Buffer or 10 µg/mL ALT, the electrodes were transferred to 1 mM solution of Fe(CN)₆ ^4-/3- in 0.1 M NaClO₄ for the EIS measurements.

Figure 7. Specificity of the ALT biosensor.

The shift in QCM frequency in response to injections of SA, BSA or ALT on the Au-Pep-Cys modified crystal. The concentrations of all three solutions were 10 µg/mL.

Figure 8. Response curves of ALT binding.

Measurements were made using QCM (shown as absolute value of frequency change) (A) and EIS (B). Each run was performed using a single electrode with successive tests in ALT solutions from low to high concentration. The apparent amount of bound ALT (N_ALT) for the QCM measurements was calculated using Eqn. 3 and is shown on the right ordinate. Error bars represent standard deviations obtained from triplicate measurements.