To verify the successful construction of the desired all-in-one test strip, the fabrication process was carefully characterized by several means. First, the self-assembly process of TMB and hemin with the help of BSA was featured by UV–vis. As depicted in Fig. 1B, the characteristic peaks of free TMB (287 nm), hemin (385 nm) and BSA (280 nm) are clearly observed in the collected TMB-hemin-BSA, and the Soret band of hemin (385 nm) in the TMB-Hemin-BSA assembly undergoes a slight bathochromic shift (Bai et al., 2011; Hua et al., 2021; Wang et al., 2017). Both the phenomena reveal the successful conjugation of TMB, hemin, and BSA. The SEM proof (Fig. 1C) verifies that the TMB-hemin-BSA assembly has a one-dimensional (1D) rod-like morphology, with an average length of ∼15 μm and an average width of ∼1 μm (Fig. S1, Supporting Information). The formation of such TMB-hemin-BSA rods is mainly driven by non-covalent interactions among BSA, porphyrin-like hemin and aromatic TMB, including aromatic π-π stacking, hydrogen bonding, and hydrophobic interactions (Bai et al., 2011). Meanwhile, the elemental mapping result (Fig. S2, Supporting Information) implies a homogeneous distribution of C, N, O, S and Fe in these TMB-hemin-BSA rods. In addition, the FTIR spectra (Fig. S3, Supporting Information) present some distinguishable adsorption bands of TMB-hemin-BSA, which can well be attributed to the three precursors (TMB, hemin, and BSA). Typically, the peak at 1680 cm−1 belongs to the C[dbnd]O stretching vibration of BSA, the peaks at 1473, 1619, 3424 and 3355 cm−1 are assigned to the C–N =, C[dbnd]C and N–H stretching vibrations of TMB, respectively, and the peaks at 933 and 1704 cm−1 belong to the O–H out-of-plane bending and C[dbnd]O stretching vibrations of hemin, respectively (Sun et al., 2021; Zhu et al., 2022b).Since the immobilization of AChE and ChO was based on electrostatic interactions, the charge changes during the self-assembly process were investigated. As compared in Fig. 1D, the formed TMB-hemin-BSA is negatively charged. After incubation with the cation polymer PEI, the zeta potential of TMB-hemin-BSA changes from −28.5 mV to 13.71 mV. When negatively charged AChE and ChO in neutral solution are further self-assembled onto the TMB-hemin-BSA surface, the zeta potential turns to be negative again (−10.9 mV), indicating that the bioenzymes AChE and ChO have been immobilized onto TMB-hemin-BSA with the help of PEI via electrostatic interactions. As seen in Fig. 1E, the self-assembly process of AChE and ChO does not destroy the shape of TMB-hemin-BSA, and the TMB-hemin-BSA-AChE-ChO assembly remains a rod-like structure. The FTIR spectra (Fig. S3, Supporting Information) were also used to certify the self-assembly process of enzymes and TMB-hemin-BSA. Typically, the new band presented at 1580 cm−1 is associated with the N–H bending vibration of PEI and the enzyme amide II band, which also indicates the existence of AChE and ChO in the all-in-one assembly (Chauhan and Pundir, 2011; Wan et al., 2019). Furthermore, FITC-labelled AChE and RhB-labelled ChO are observed in the laser confocal fluorescence microscopy images (Fig. 1F), also demonstrating the successful immobilization of the two bioenzymes onto TMB-hemin-BSA. As checked by XPS (Fig. 1G), signals assigned to C, N, S and Fe are found in the TMB-hemin-BSA-AChE-ChO all-in-one assembly as expected.Considering that several cascade reactions occur in the test strip, here several critical parameters possibly affecting the catalytic/sensing efficiency were systematically studied. The signal amplification module consisting of TMB, hemin and BSA was first investigated. As found in Fig. 2C, with the amount of BSA increases, the formed TMB-hemin-BSA rods exhibit a better dispersibility in aqueous solution. When 90 mg BSA is employed to fabricate the TMB-hemin-BSA assembly, no obvious precipitate is observed. Such an excellent dispersion of TMB-hemin-BSA can facilitate the subsequent bioenzyme loading process. Then, the ratio of chromogenic substrate TMB to peroxidase-like hemin was also studied. When the ratio is adjusted from 1:0.1 to 1:7, the absorbance of the TMB-hemin-BSA + H2O2 solution shows a volcano-type trend, and the same change trend is also observed in solution (Fig. 2D). When a ratio of 1:0.6 is applied as the optimal parameter, according to the standard curves (Fig. S4, Supporting Information), the loading capacities of TMB and hemin are estimated to be 598.6 and 22.3 mg g−1, respectively. Such a high chromogen loading can ensure the generation of remarkable signals for sensitive detection (Khoris et al., 2021). As for the effect of buffer pH, similar to natural peroxidase (Niu et al., 2016), the TMB-hemin-BSA assembly exhibits a pH-dependent catalytic activity (Fig. 2E), with the optimal activity at pH 5.0. As expected, when more TMB-hemin-BSA rods are used to trigger the chromogenic reaction, the observed absorbance also increases (Fig. 2F). With a remarkable absorbance value as well as the minimum background color, the amount of the TMB-hemin-BSA assembly is set as 0.035 mg. By taking the utilization of bioenzymes and their immobilization efficiency into consideration, the usages of AChE and ChO were also evaluated in solution. As presented in Fig. 2G and H, an absorbance growth trend is first found with the increase of AChE or ChO amount, followed by an equilibrium phenomenon. Accordingly, the optimal dosages of AChE and ChO are set as 0.01 U and 0.35 U, respectively.More importantly, our all-in-one test strip is able to offer an enhanced cascade catalytic efficiency. In the present study the catalytic efficiencies of the all-in-one assembly and TMB + hemin + free AChE + free ChO were carefully compared. As found in Fig. S5 (Supporting Information), an enhanced UV–vis signal attributed to the oxidation of TMB is observed in the all-in-one assembly compared to the counterpart. As a result, a 1.22-fold improvement of the cascade catalytic efficiency is obtained in the all-in-one assembly (Fig. 3C). Such a higher efficiency is mainly attributed to the proximity effect of all cascade reactions occurring on the all-in-one assembly, which can minimize the diffusion and decomposition of reactants and intermediates, thus improving the efficiency of chromogenic assays. The higher cascade catalytic efficiency will benefit the highly sensitive detection of targets.To realize the high-performance sensing of pesticides, the total usage of the two bioenzymes (AChE and ChO) for electrostatic self-assembly was further optimized. As the total usage of AChE and ChO increases, more bioenzymes are immobilized, and more H2O2 is produced to result in a more significant color change (Fig. 3D and Fig. S6, Supporting Information). However, the absorbance difference before and after adding paraoxon undergoes a volcano-type change (Fig. 3D), indicating that excess enzymes can lead to the reduction in the sensitivity of pesticide detection. As a result, the optimal ratio of bioenzymes to TMB-hemin-BSA is set as 1.03:1. In addition, the immobilization efficiency of bioenzymes at the optimal ratio was determined. After the co-immobilization of AChE and ChO onto TMB-hemin-BSA rods, the relative fluorescence intensities of labelled AChE and ChO remaining in solution decrease (Fig. S7, Supporting Information), and the immobilization efficiency of AChE and ChO is calculated to be 40.3% and 83.2%, respectively.The authors appreciate the supports from the Faculty of Agricultural Equipment of Jiangsu University (NZXB20210207), the National Natural Science Foundation of China (21605061), the State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology (ES202122), and the Jiangsu Provincial Key Laboratory of Environmental Science and Engineering (JSHJZDSYS-202101). X. Niu also appreciates the Star-up Research Fund from University of South China.
