SpyDirect: A Novel Biofunctionalization Method for High Stability and Longevity of Electronic Biosensors

Abstract

Electronic immunosensors are indispensable tools for diagnostics, particularly in scenarios demanding immediate results. Conventionally, these sensors rely on the chemical immobilization of antibodies onto electrodes. However, globular proteins tend to adsorb and unfold on these surfaces. Therefore, self-assembled monolayers (SAMs) of thiolated alkyl molecules are commonly used for indirect gold-antibody coupling. Here, a limitation associated with SAMs is revealed, wherein they curtail the longevity of protein sensors, particularly when integrated into the state-of-the-art transducer of organic bioelectronics-the organic electrochemical transistor. The SpyDirect method is introduced, generating an ultrahigh-density array of oriented nanobody receptors stably linked to the gold electrode without any SAMs. It is accomplished by directly coupling cysteine-terminated and orientation-optimized spyTag peptides, onto which nanobody-spyCatcher fusion proteins are autocatalytically attached, yielding a dense and uniform biorecognition layer. The structure-guided design optimizes the conformation and packing of flexibly tethered nanobodies. This biolayer enhances shelf-life and reduces background noise in various complex media. SpyDirect functionalization is faster and easier than SAM-based methods and does not necessitate organic solvents, rendering the sensors eco-friendly, accessible, and amenable to scalability. SpyDirect represents a broadly applicable biofunctionalization method for enhancing the cost-effectiveness, sustainability, and longevity of electronic biosensors, all without compromising sensitivity.

Keywords: cysteine‐peptide linker; nanobody; organic bioelectronics; organic electrochemical transistor; protein sensor; self‐assembled monolayer.

Figure 1

Covalent nanobody conjugation via self‐catalyzing spyTag/spyCatcher bond. A) SAM‐based nanobody immobilization. A synthetic spyTag peptide (marked in a red box) is chemically coupled to the HDT monolayer via maleimide click chemistry. The nanobody‐spyCatcher fusion protein attaches itself to this chemical layer through the autocatalytic formation of a covalent spyTag‐spyCatcher bond. B, C) SpyDirect‐based nanobody immobilization. Direct biofunctionalization of the Au gate electrode with a cysteine‐terminated spyTag‐peptide followed by the coupling of the nanobody‐spyCatcher fusion protein. (B) SpyDirect N′ coupling. The SpyTag is immobilized through an N‐terminal cysteine. (C) SpyDirect C′ coupling. The inverted SpyTag immobilization through a C‐terminal cysteine removes steric constraints for SpyCatcher packing and linker‐nanobody arrangement.

Figure 2

Comparison between the HDT SAM‐based and the SpyDirect‐based nanobody (VHH72) surface. A) QCM‐D profile of the coupling of (1) the spyTag‐peptide (maleimide‐modified peptide versus cysteine‐terminated peptide), (2) VHH72‐spyCatcher fusion protein, (3) bovine serum albumin (BSA), and binding of (4) non‐target GFP and (5) SARS‐CoV‐2 spike protein. HDT SAM‐based nanobody experiment uses an HDT‐coated gold QCM‐D sensor, while SpyDirect uses a bare gold QCM‐D sensor. All the mass was calculated in PBS (10 mm, pH 7.4). Atomic force microscopy (AFM) characterization of B) HDT SAM‐ and C) SpyDirect‐based biofunctional electrode surface in PBS (10 mm, pH 7.4). No BSA was added. A computational rule‐based model generated from the QCM‐D results depicting the D) HDT SAM‐ and E) SpyDirect‐based biofunctional electrode surfaces (to scale).

Figure 3

Physiochemical and electrochemical characterization of SpyDirect‐based nanobody functionalized Au electrode. A) High‐resolution XPS spectra for Au 4f, C 1s, and N 1s of the gold surface after immobilization of cysteine‐terminated spyTag‐peptide and the VHH72‐spyCatcher. The VHH72‐spyCatcher buffer contained BSA. B) Cyclic voltammogram, C) Bode plot (solid lines and dotted lines correspond to the magnitude and the phase of the impedance, respectively), D) Nyquist plot of the gold electrode before and after the subsequent functionalization with cysteine‐terminated spyTag peptide, and the VHH72‐spyCatcher. Inset to D is the equivalent circuit model used to fit the impedance spectra. E) The R _ct and C _dl of the electrode. The measurements were done in 10 mm [Fe(CN)₆]^3−/4− in 10 mm PBS, pH 7.4.

Figure 4

Detection of SARS‐CoV‐2 spike protein in saliva and untreated wastewater. A) Sensor operation. B, C) The response of SpyDirect‐based OECT sensors (nanobody is VHH72) to random concentrations of SARS‐CoV‐2 spike protein in raw saliva and untreated wastewater. D) Background response of SpyDirect‐ and HDT SAM‐based sensors (nanobody is VHH72) to human saliva, UTM, and untreated wastewater. GFP nanobody‐functionalized gate electrodes were used as a control.

Figure 5

Stability tests of VHH72‐biofunctionalized gates. The normalized response of OECTs to SARS‐CoV‐2 spike protein (S1) (1.2 pm, in saliva) when gated with A) HDT SAM‐based electrodes and B) SpyDirect‐based electrodes. The biofunctionalized gates were stored in 10 mm PBS, pH 74 at 4 °C for 3, 7, and 14 days before use.

Figure 6

A) The charge transfer resistance (R_ct) of SpyDirect‐ and HDT SAM‐based gates. The impedance measurements were performed with 10 mm [Fe(CN)₆]^3−/4− in 10 mm PBS, pH 7.4. The Randles circuit model was used to fit the impedance spectra. The biofunctionalized gates were stored in 10 mm PBS, pH 7.4 at 4 °C for 3, 7, and 14 days before use. XPS spectra of C 1s, O 1s, and N 1s of B) SpyDirect‐ and C) HDT SAM‐based gates before and after 7‐day storage in 10 mm PBS, pH 7.4 at 4 °C. D) The response of OECTs gated with SpyDirect‐based electrodes prepared from dried cysteine peptide. The peptide‐modified gates were stored for 7 days in ambient conditions before the nanobody immobilization. All electrode and sensor measurements were done with at least three electrodes.