Highly stable and reusable imprinted artificial antibody used for in situ detection and disinfection of pathogens

We fabricate artificial antibodies based on imprinting technology and develop a sandwich ELISA for pathogen detection.


Introduction
The enzyme-linked immunosorbent assay (ELISA) is the current gold standard for clinical biomarker detection, as well as a robust approach for pathogen screening. Arguably, the most powerful ELISA format is the sandwich assay, [1][2][3] in which the antigen is recognized by a couple of antibodies: a capture antibody (cAb) and detection antibody (dAb). The two antibodies recognition strategy endues the detection with high sensitivity and specicity. [4][5][6] However, the main drawbacks of the sandwich ELISA are the cost, and the labor-intensive and time-consuming procedure for the screening of antibodies. In addition, owing to their easily disrupted stabilities, the natural antibodies usually cannot be reused. Furthermore, an appropriate enzyme should be linked to the dAb to clearly show the detection results. The complex process and expensive probes mean that the sandwich ELISA is not an optimal method for high-throughput screening of real samples. Therefore, the design and synthesis of articial antibodies with easy availability and high stability, as alternatives to the natural antibodies, is urgently in demand for biodiagnostic applications.
Molecular imprinting technology (MIT) has been identied as a promising approach to synthesize articial antibodies. [7][8][9] The synthesized articial antibodies exhibit a natural antibody-like binding affinity and selectivity. Intriguingly, they can even possess better characteristics than natural antibodies, including easy availability and operability, high stability to harsh chemical and physical conditions, and some even have superior reusability. To date, articial antibodies against low molecular weight compounds 10-12 and biological macromolecules 13-16 based on MIT have been employed for a myriad of applications, such as separation, 17 biomimetic catalysis, 18 sensing, [19][20][21][22] sewage treatment, 23-25 enzyme inhibition [26][27][28] and so on. In spite of promising prospects for molecular imprinting, it becomes more challenging as the target size increases, although nanoparticles 29 and bioentities, such as viruses, 30-32 microbes 33-38 and mammalian cells, 39,40 as templates for articial antibody fabrication have recently been reported.
Herein, for the rst time, we demonstrate a cell imprinted articial antibodies-based sandwich ELISA for pathogen detection. Both the cAbs and dAbs were synthesized via an imprinting procedure. The cAbs were in situ fabricated on an indium tin oxide (ITO) conductive glass surface through an electrochemically assisted polycondensation method. 41,42 The dAbs were synthesized using a sol-gel method with cerium dioxide nanoparticles (CeO 2 NPs) integrated as articial nanoenzymes. CeO 2 NPs have recently been reported to possess excellent peroxidase-like activity toward the substrate 3,3 0 ,5,5 0tetramethylbenzidine (TMB), [43][44][45] which can be used to fabricate immunoassays. 46,47 With the properties of easy availability, and superior stability and reusability, the fabricated articial antibodies may circumvent the limitations of the natural antibodies and maintain natural antibody-like binding affinities and selectivities. What's more, with their conductivity properties, the cAbs can even disinfect the captured pathogen in situ by using an electrochemical technique.

CAb fabrication
As illustrated in Fig. 1A, the cAbs were fabricated on an ITO glass surface using Staphylococcus aureus (S. aureus) as a model of the target pathogen, which is one of the ve most common causes of nosocomial infections. S. aureus was rst immobilized on the aldehyde functionalized ITO glass surface through a Schiff base linkage ( Fig. S1A and S1B †). Subsequently, a silica lm was deposited on the electrode surface around the S. aureus, via an in situ electrochemically assisted polycondensation method (Fig. S1C †). 41,42 Finally, the cAbs were obtained through a calcination treatment. Aer removal of the template, many regular cavity-cAbs were found to be scattered on the surface of the ITO glass ( Fig. 1B and C). Images of the cavities at a higher magnication revealed their circular shape ( Fig. 1B and C). The depths of the cavities were measured to be about 160 nm from the AFM image (Fig. 1C). Evidently, the three-dimensional spheroidal architecture of the template pathogen was imprinted well on the ITO glass surface. The fabrication procedure was also characterized using electrochemical methods (Fig. S2 †).

DAbs fabrication
The enzyme-linked dAbs were obtained through four simple steps: (i) in situ encapsulation of S. aureus with a silica shell, (ii) deposition of CeO 2 NPs on the silica shell surface, (iii) calcination to remove the template and (iv) ultrasonic treatment to crush the hollow SiO 2 @CeO 2 shells ( Fig. 2A and S4 †). Fig. 2B shows a typical TEM image of the hollow SiO 2 @CeO 2 shells aer removal of the template pathogen. The cavities of the hollow spheres were similar in size to S. aureus. Meanwhile, a thin CeO 2 shell was found to be uniformly deposited on the hollow sphere surface ( Fig. 2B and C). Aer a harsh ultrasonic treatment, the hollow spheres were cracked and cap-like dAbs were obtained (Fig. 2D). The empty cavities of the dAbs were found to maintain the size and shape of the original S. aureus. Altogether, these results conrmed that the shape and size of the template pathogen were preserved.
The enzyme that is linked to the dAb is critical for the assay because it will directly catalyze reaction of the substrate to produce a detectable signal. In the present work, CeO 2 NPs were chosen as articial nanoenzymes and integrated with the dAb. The oxidation of TMB by CeO 2 NPs in the presence of H 2 O 2 produced a blue color, with two absorbance bands at 370 and 652 nm. 43 Fig. 2E exhibits the time-dependent absorbance change (at 652 nm) for different concentrations of the dAbs. The dAbs demonstrated a high catalytic activity toward the oxidation of TMB. Notably, even 2 mg mL À1 of the antibodies could produce a detectable signal within 10 min (Fig. 2E).

Target pathogen recognition tests
Having successfully fabricated both the cAbs and dAbs, we next investigated the target pathogen recognition capacity of these  antibodies. As shown in Fig. 3A, S. aureus was only found to be present on the imprinted cavities, and none was found in the non-imprinting area, indicating that S. aureus could be efficiently captured by the cAbs. Meanwhile, the non-target pathogens Escherichia coli (E. coli) and yeast cells were difficult to nd on the plate (Fig. S5 †). Even Staphylococcus epidermidis (S. epidermidis), which is similar in shape and size to S. aureus, was found to be much less captured by the cAbs (Fig. S7 †). The recognition capacity of the dAbs was characterized using uorescence microscopy. For better identication, S. aureus was stained with calcein-AM, to give a green uorescence. Before photographing, the stained S. aureus and dAbs were dispersed in buffer and then moderately shaken for 10 min. As shown in Fig. 3B-D, most of the green uorescence and crescent-shaped dAbs overlapped at the same spot, revealing that the dAbs were tightly bound to S. aureus. The binding specicity of the dAbs was also evaluated. Evidently, the dAbs could specically bind to S. aureus over E. coli, yeast cells and S. epidermidis (Fig. S6 and S7 †). Taken together, both the fabricated cAbs and dAbs could recognize target pathogens with high specicity. The target pathogen-like size and shape of the imprinted cavities may play an important role for the high recognition and selectivity capability. 35,36,40 In addition, the imprinting of the surface chemistry of the pathogen at the molecular level would also contribute to the natural antibody-like properties. 33,34,40 Pathogen detection The target pathogen recognition capability of both the cAbs and dAbs, and the high catalytic activity of the dAbs, adequately meet the requirements for construction of a sandwich ELISA. Therefore, a sandwich ELISA based on these articial antibodies was set up for S. aureus detection. A schematic representation of the sandwich ELISA format is presented in Fig. 4A. The target pathogens were rst selectively captured by the cAbs-functionalized ITO glass, and subsequently the captured pathogens were recognized by the dAbs. Finally, the blue color was generated through the oxidation of TMB by the dAbs in the presence of H 2 O 2 . The constructed assay was characterized using SEM imaging. As shown in Fig. 4B, the sandwich structure was clearly observed, showing that S. aureus was captured by the cAbs and capped by the dAbs. The detection results are illustrated in Fig. 4C. No visible color was found on the control plate, while a distinguishable blue color appeared in the presence of S. aureus at 10 4 CFU mL À1 (colony-forming units per milliliter). The color deepened gradually as the S. aureus concentration increased. UV-vis absorption spectra were used to quantify the results (Fig. 4D). The limit of detection was estimated to be about 500 CFU mL À1 , which is much lower than that of traditional ELISA methods (10 4 to 10 5 CFU mL À1 ). 48 The detection results could also be analyzed using image processing soware (Adobe Photoshop) 49 (Fig. S8 †). The selectivity of the constructed sandwich ELISA was further investigated using non-target pathogens, E. coli, yeast cells and S. epidermidis, as controls. As presented in Fig. 4C, no obvious blue color appeared for the non-target pathogen detections, indicating the high specicity of the fabricated assay. The specic detection capability could be ascribed to the recognition specicity of the articial antibodies. In addition, the two antibodies recognition strategy also plays an active role.

Reusability tests
The reusability of natural antibodies is severely hampered by their poor stability. Herein, the articial antibodies synthesized  went through a high temperature calcination procedure, and they all exhibited high thermal stability. More importantly, both the cAbs and dAbs were found to be reusable through a simple calcination treatment. Aer calcination, the cAbs were found to maintain their original topography (Fig. S9 †) and no obvious attenuation of the catalytic activity of the dAbs was observed (Fig. S10 †). Even aer treatment three times, the recovered sandwich ELISA could give a detection signal as high as 90% of the initial value (Fig. 4E), indicating their high reusability.

Pathogen electrochemical disinfection
For healthcare, it is more desirable that the detection and disinfection of pathogens can be realized at the same time. The fabricated cAbs could not only be used to construct a sandwich ELSA for pathogen detection, but could also be available to in situ electrochemically disinfect the captured pathogens. The uorescent probes calcein-AM and propidium iodide (PI) were used to stain the living cells (green) and dead cells (red), respectively. As shown in Fig. 5A and B, aer electrochemical treatment almost all of the S. aureus could be stained by PI, indicating that the S. aureus was disinfected. The electrochemical oxidation of intracellular coenzyme A (CoA), which is irreversibly converted to a CoA dimer by disulde bond formation, and disruption of cell membranes have been generally considered to be responsible for disinfection activities. 50,51 The cell membrane disruption effect was further demonstrated through SEM imaging. As seen in Fig. 5C, the morphology of the S. aureus was remarkably disrupted; many cells were found to be greatly shrunken and some had even collapsed.
Taken together, the fabricated articial antibodies based on imprinting technology can be used for facile construction of a sandwich ELISA for the sensitive detection of pathogens. Compared with recently reported assays for the detection of pathogens (Table S1 †), the most obvious advantage of the present method is that all the construction components are integrative, stable and reusable. In addition, apart from detection, the captured pathogens can even be in situ electrochemically disinfected.

Conclusions
In summary, for the rst time we have fabricated cell imprinted articial antibodies to set up a sandwich ELISA for pathogen detection. Both the cAbs and dAbs were obtained via in situ methods, with simplicity, rapidity and low cost. The fabricated antibodies could be used without immobilization or an enzyme linkage procedure, which would streamline the process of sandwich ELISA construction. The constructed ELISA could be used for target pathogen detection with high sensitivity and selectivity. What's more, these articial antibodies possess superior stability and reusability, which may circumvent the limitations of the natural antibodies. Besides, the cAbs can disinfect pathogens in situ by using an electrochemical technique. Thus, the present work may open a new avenue for designing stable and reusable articial antibodies for immunoassays.