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.

and dissipation (ΔD) are plotted over time as (1) spyTag-cysteine, (2) GFP nanobody-spyCatcher was introduced to the system, followed by washing steps.Atomic force microscopy (AFM) was used to confirm the change in the surface roughness and feature height during biofunctionalization.Before immobilizing any biomolecules, the root mean square (RMS) roughness of the electrode is 4.5 nm, and the mean height of the grains is 14.7 nm.
After incubating with the nanobody solution, large particles were observed on the gate electrode surface.The RMS roughness of the nanobody-modified electrodes increased to 6.0 nm.The mean feature height increased by 8.4 nm (from 14.7 to 23.1 nm), verifying the successful immobilization of nanobody and BSA.   Figure S6a shows that at low VD, the increase in ID is significant, followed by a saturation regime at higher VD, consistent with accumulation mode OECT operation.The device showed minimal hysteresis with almost identical behavior, as observed from forward and backward voltage scans (Figure S6b).The p(g0T2-g6T2) transistors had low OFF-currents on the order of 10 μA, and an ON/OFF ratio of up to 100 at VG, which led to maximum gm in the saturation regime (Figure S6b).
Our OECT had a lower power demand (75 μW at VG = -0.05V, VD = -0.1 V) when operated at the subthreshold regime, which yielded the maximum sensor NR values (Figure S6c).We investigated the operational stability of our devices by switching them "ON" and "OFF" for 10 s (VG = -0.5Vor 0 V) each at constant VD= -0.5 V and recording the ID over 360 cycles performed within 2 hours (Figure S6d).The device retained 98% of its initial current after cycling.Small gate voltages applied to keep the device in its ON-state reduce the risk of material instability for long-term use requirements.

Figure S1 .
Figure S1.QCM-D monitoring of the GFP nanobody immobilization through coupling with (a) spyDirect N' or (b) spyDirect C'.The QCM-D signals including the change in frequency (Δf)

Figure S4 .
Figure S4.Relative distribution of feature heights of nanobody functionalized surfaces prepared through the two methods (HDT SAM vs. SpyDirect) determined using Gaussian fitting.

Figure S5 .
Figure S5.Optical picture of one chip comprising 6 channels (100 µm × 10 µm) covered with ptype material p(g0T2-g6T2) film prepared by spin-coating.The polymer-coated microelectrodes shown in the image were used for the electrochemical characterization of the material.

Figure S6 .
Figure S6.(a) The output characteristics of p(g0T2-g6T2) devices with VG varying from 0.2 V to -0.6 V.The arrows indicate the scan direction of VG.The scan rate was 50 mV/s.(b) Ten repetitive transfer curves of exemplary devices recorded at VD = -0.1 V.The arrows in the inset figure indicate the scan direction hysteresis for the last repetition.(c) The calculated power consumption at different VG (VD = -0.1 V).(d) Transient characteristics of z p(g0T2-g6T2) device over an hour of continuous ON and OFF biasing (10 seconds each) at the gate electrode.The operation conditions are VD = -0.5 V, VG = -0.5 V (or 0 V).All measurements were performed in 10 mM PBS using an Ag/AgCl as the gate electrode.

Figure S7 .
Figure S7.The output characteristics obtained by sweeping VD from 0 to -0.4 V and VG from 0.2 V to -0.4 V in PB (40 mM, pH 7.4).(a) The first I-V and (b) the 500 th I-V curves.The change of channel current at VD = VG = -0.4V is 0.8%.All measurements were performed in PB (40 mM, pH 7.4) using an Ag/AgCl as the gate electrode.

Figure S9 .
Figure S9.The electrochemical capacitance of a VHH72 electrode upon protein binding.The EIS measurements were performed in 40 mM PB, pH 7.4.The data were fitted using Randles circuit to extract the capacitance.Lysozyme was used as a negative control.