A Single Aptamer-Dependent Sandwich-Type Biosensor for the Colorimetric Detection of Cancer Cells via Direct Coordinately Binding of Bare Bimetallic Metal–Organic Framework-Based Nanozymes

A typical colorimetric sandwich-type sensor relies on dual antibodies/aptamers to specifically visualize the targets. The requirement of dual antibodies/aptamers and low signal intensity inevitably increases the design difficulty and compromises the sensing sensitivity. In this work, a novel sandwich-type aptasensor was developed using single aptamer-functionalized magnetic nanoparticles as a specific recognition unit to target cancer cells and a bimetallic metal–organic frameworks (MOFs)-based nanozymes as a colorimetric signal amplification unit. The well-defined crystalline structure of UIO-66 MOFs enabled the introduction of Fe/Zr bimetal nodes, which possessed integrated properties of the peroxidase-like nanozyme activity and direct coordinately binding to the cell surface. Such a novel construction strategy of sandwich-type aptasensors achieved simple, sensitive, and specific detection of the target cancer cells, which will inspire the development of biosensors.


Introduction
Cancer remains a major threaten to human life in the world [1]. Current clinical practice suggests that early detection of cancer cells contributes to monitoring the progressions of cancer and conducting precise therapy [2]. Consequently, it is desirable to develop simple, sensitive, and specific technologies to quantify cancer cells. Among all the developed detection strategies, colorimetric sandwich-type methods have attracted considerable attention due to the advantages of high specificity, easy operation, as well as bare-eye-based readout [3]. In general, the colorimetric sandwich-type assay is carried out by using two specific antibodies/aptamers to recognize the targets and using appropriate labels to provide detectable signals [4,5]. However, the requirement of two antibodies/aptamers inevitably increases the design difficulty. In fact, the use of single high-affinity antibody/aptamer fully meets the need of specific recognition of target cells [6][7][8]. In addition, low colorimetric signals greatly compromise the response sensitivity of visual analysis.
Metal-organic frameworks (MOFs) are a type of highly ordered porous crystalline material constructed by reasonable self-assembly of metal ions and organic ligands [9,10]. The well-defined crystalline structure of MOFs offers an excellent alternative to design metal nodes to acquire metal-related physicochemical properties, which holds great potential in colorimetric sandwich-type analysis. For instance, the introduction of Fe atoms might endow the resulting Fe-containing MOFs with intrinsic peroxidase-like catalytic activity [11], which could significantly increase the colorimetric analysis sensitivity via accelerating H 2 O 2 -mediated oxidation of chromogenic substrates. The coordination affinity between Zr 4+ and PO 4 3− contributes to identifying the cellular phospholipid bilayer using Zr-containing MOFs [12]. Correspondingly, developing bimetallic MOFs with a given ionic ratio offers an interesting candidate to obtain integrated properties from different metal nodes.
Herein, a novel sandwich-type aptasensor was prepared for colorimetric analysis of cancer cells using single aptamer-modified magnetic nanoparticles to selectively capture and separate target cells, followed by direct binding of bare bimetallic metal-organicframework-based nanozymes. By adjusting the added amount of metal precursors among the synthesis of a given UIO-66 MOF, UIO-66(Fe/Zr) bimetallic MOFs were prepared with integrated properties, including the peroxidase-like nanozyme activity from Fe nodes and direct binding to cell surface based on the Zr-involved coordination bond. Such a single aptamer-dependent colorimetric sandwich-type aptasensor facilitated simple, sensitive, and specific detection of target cancer cells even in complex biological systems.

Preparation of UIO-66(Fe/Zr)
The UIO-66(Fe/Zr) NPs were synthesized following a previously reported method [13]. In brief, 0.8 mM ZrCl 4 , 0.8 mM FeCl 3 ·6H 2 O, and 0.8 mM H 2 BDC were mixed into 25 mL of DMF, followed by the addition of 5 mL of acetic acid. After ultrasonic treatment at ambient temperature, the mixture was transferred to a 50-mL Teflon-lined hydrothermal reactor and reacted at 120 • C for 12 h. Subsequently, the obtained products were washed with ultrapure water and ethanol three times, respectively. After drying the products at 60 • C overnight, UIO-66(Fe/Zr) powder was collected for further use. For the preparation of Cy3-dye-incorporated UIO-66(Fe/Zr), 9.7 mg of UIO-66(Fe/Zr) NPs were mixed with 0.03 mM Cy3 dyes, followed by stirring for 24 h.

Preparation of AS1411-Functionalized Fe 3 O 4 @SiO 2
First, Fe 3 O 4 @SiO 2 core-shell NPs were synthesized using a reported sol-gel strategy [14]. Typically, 0.15 g of Fe 3 O 4 NPs were ultrasonically dispersed into a mixture solution containing 70 mL of ultrapure water, 280 mL of ethanol, and 5.0 mL of NH 3 ·H 2 O (28 wt%), followed by a slow addition of 4.0 mL of TEOS. After stirring for 10 h, the resulting Fe 3 O 4 @SiO 2 NPs were collected after magnetic separation and washing with ultrapure water and ethanol.

Cell Culture
Human cervical cancer HeLa cells were cultured in 1640 medium containing 10% FBS and 1% penicillin-streptomycin. For the cases of human hepatoma HepG2 cells and mouse fibroblast L929 cells, DMEM medium was used. All the cell lines were incubated in a humidified atmosphere of 5% CO 2 at 37 • C.

Principle of Colorimetric Sandwich-Type Detection of Cancer Cells
As shown in Figure 1A, UIO-66(Fe/Zr) NPs were synthesized by a one-step hydrothermal method using ZrCl 4 and FeCl 3 ·6H 2 O as metal precursors and H 2 BDC as organic ligands. In order to guarantee selective detection of target cancer cells, a novel sandwich-type aptasensor was proposed using aptamer-functionalized Fe 3 O 4 @SiO 2 core-shell NPs to selectively capture and magnetically separate cancer cells from complex biological systems ( Figure 1B). Considering bare Fe 3 O 4 NPs also own peroxide-like nanozyme activity, Fe 3 O 4 @SiO 2 core-shell NPs originating from the silylanization of Fe 3 O 4 NPs were adopted here to improve the signal-to-noise ratio toward cancer cell analysis. Taking advantage of the Zr-O-P coordination bond between phosphate units in the phospholipid bilayer of the cell membrane and zirconium nodes on MOFs, UIO-66(Fe/Zr) could directly bind to the cell surface. In addition, UIO-66(Fe/Zr) possesses a strong peroxidase-like nanozyme activity, which can catalyze H 2 O 2 -mediated oxidation of colorless TMB to form yellow-colored oxidized ones (oxTMB) with the aid of 1 M H 2 SO 4 , providing an excellent alternative for colorimetric analysis of cancer cells.  Figure 2E) [17]. These results indicated that the introduction of additional Fe nodes was unable to disturb the intact metal-organic framework of UIO-66(Zr), consistent with that reported previously [13]. Taken all the above results into consideration, it can be concluded that a part of the Zr nodes in the UIO-66 framework were carefully displaced by Fe nodes, and the similar Fe-O-Fe bond to Zr-O-Zr bond ensured a comparable spatial configuration of the UIO-66(Fe/Zr) to that of UIO-66(Zr). Next, Fe 3 O 4 @SiO 2 core-shell NPs were prepared via sonication-assisted coating of a silica shell on Fe 3 O 4 NPs core. As shown in Figure S1, spherical Fe 3 O 4 NPs had a mean diameter of approximately 320 nm. After being coated with a thin silica shell (approximately 32 nm), an obvious core-shell structure was observed for the obtained Fe 3 O 4 @SiO 2 NPs. Strong asymmetric stretching peaks of the Si-O-Si bond at 1207 and 1079 cm −1 appeared [18], accompanied by the disappearance of the characteristic peak of the Fe-O-Fe bond at 593 cm −1 [19], which indicated the successful coating of SiO 2 on the surface of Fe 3 O 4 NPs ( Figure S2A). For the formed Fe 3 O 4 @SiO 2 core-shell NPs, aminated treatment gave rise to a positive zeta potential ( Figure S2B). However, further functional modification with aptamer AS1411 molecules induced the resultant Fe 3 O 4 @SiO 2 -Apt NPs to be negative-charged.

Assessment of Peroxidase-Like Nanozyme Activity
The peroxidase-like nanozyme activity was visually evaluated using the typical TMB-H 2 O 2 colorimetric system [7]. As shown in Figure 3A, under the condition of the acetate buffer, the mixture of TMB and H 2 O 2 almost remained colorless with a low absorption peak intensity at 450 nm, indicating a relatively slow reaction efficiency (inset b). After introduction of UIO-66(Fe/Zr) (inset d), the solution color of the TMB+H 2 O 2 system turned to be yellow. Since UIO-66(Fe/Zr) NPs alone exhibited negligible absorption at 450 nm (inset c), the above-mentioned yellow solution was, thus, attributed to the catalytic decomposition of H 2 O 2 by UIO-66(Fe/Zr) NPs to promote the oxidation of TMB ( Figure 3B). Furthermore, the absorption peak intensity was positively related to the added amount of UIO-66(Fe/Zr) NPs ( Figure 3C), revealing the feasibility of visual analysis using a UIO-66(Fe/Zr)-mediated colorimetric system. In order to obtain a low background signal, Fe 3 O 4 @SiO 2 -Apt were expected without peroxidase-like activity. As anticipated, bare Fe 3 O 4 NPs did catalyze the decomposition of hydrogen peroxide to accelerate TMB oxidization (inset b, Figure S3). For the case of Fe 3 O 4 @SiO 2 -Apt, effective coverage of Fe 3 O 4 with a thin shell of SiO 2 resulted in a significant decrease in the number of catalytic sites, and the peroxidase-like activity was almost lost (insets c and d, Figure S3).

Construction of a Single-Aptamer-Based Sandwich-Type Biosensor
The direct binding of bare UIO-66(Fe/Zr) to target cancer cells was of vital importance for constructing a single-aptamer-based sandwich-type biosensor. To this end, Cy3-dyesincorporated UIO-66(Fe/Zr) NPs were prepared for fluorescence-assisted positioning of their binding sites ( Figure S4). As a proof-of-concept assay, we attempted to incubate human cervical cancer HeLa cells with Cy3-dyes-incorporated UIO-66(Fe/Zr) NPs. As imaged by confocal laser scanning microscopy (CLSM), the outline of the HeLa cell was clearly lighted up, taking advantage of the luminance of Cy3 ( Figure 4A), which proved the successful binding of bare UIO-66(Fe/Zr) NPs to the surface of HeLa cells. The resultant HeLa cell maintained the original morphology, which demonstrates the excellent biocompatibility of UIO-66(Fe/Zr) NPs.
Inspired by the direct binding of bare UIO-66(Fe/Zr) NPs to HeLa cells, a colorimetric sandwich-type aptasensor was then designed using Fe 3 O 4 @SiO 2 -Apt to selectively capture and magnetically separate HeLa cells through specific recognition of aptamer AS1411 to the overexpressed nucleolin [20,21]. To achieve the colorimetric detection of HeLa cells, Fe 3 O 4 @SiO 2 -Apt were first incubated with HeLa cells at 37 • C for 2 h, followed by the addition of UIO-66(Fe/Zr) NPs and incubation at 37 • C for another 30 min. After magnetic separation of the free nanozymes, the formed sandwich-type structure was added into the TMB+H 2 O 2 system. Without HeLa cells or UIO-66(Fe/Zr), the solution color of TMB+H 2 O 2 system in the presence of Fe 3 O 4 @SiO 2 -Apt was nearly colorless, and weak absorption at 450 nm was generated (curves a and b, Figure 4B). For the colorimetric system treated with a stable sandwich-type structure (curve c, Figure 4B), the yellow solution appeared with an over 7-fold absorbance intensity compared to that of the blank sample (inset c, Figure 4B), which demonstrates the feasibility of colorimetric detection of HeLa cells.

Colorimetric Detection of Cancer Cells
As the concentration of HeLa cells increased from 0 to 2 × 10 4 cells/mL, the colorimetric system gradually darkened in solution color (the inset, Figure 5A). By measuring the absorption intensity at 450 nm, quantitative analysis further confirmed a positive correlation between HeLa cell concentration and the oxidation efficiency of TMB ( Figure 5A). These results were originated from the fact that the presence of more HeLa cells meant more cell membrane-bound UIO-66(Fe/Zr) nanozymes, resulting in a higher catalytic reaction dynamic of TMB oxidation by H 2 O 2 . As illustrated in Figure 5B, a good linear relationship was attained between the absorption intensity at 450 nm and the cell concentration ranging from 10 3 to 10 4 cells/mL. The detection limit (LOD) was calculated to be 481 cells/mL based on 3σ/k (σ and k representing the standard deviation of blank signal and the curve slope, respectively).
To further verify the sensing selectivity toward HeLa cells, a same concentration of human hepatoma HepG2 cells and mouse fibroblast L929 cells were tested with the proposed colorimetric sandwich-type aptasensor, respectively. When compared with the blank sample, the group of L929 cells caused no apparent color change, while a significant yellow solution was produced for the samples of HepG2 and HeLa cells ( Figure 5C). Higher response sensitivity toward cancer cells might be ascribed to a higher expression level of nucleolin on cancer cells than that on normal cells [8], which verifies the sensing specificity of the colorimetric sandwich-type aptasensor. Furthermore, this aptasensor could accurately distinguish HeLa cells from an analogue biological environment via mixing HeLa and L929 cells (light-blue column, Figure 5C).
Encouraged by the excellent selectivity, the developed aptasensor was employed for the analysis of real samples by spiking different concentrations of HeLa cells into 1000-fold diluted healthy human blood. A satisfactory recovery rate between 96.8% and 106.0% with an acceptable relative standard deviation (RSD) value was obtained ( Figure 5D), which fully demonstrated the accuracy and reliability of the developed aptasensor in analyzing target cancer cells in the complex biological system.

Conclusions
In summary, a novel single aptamer-dependent sandwich-type biosensor was proposed for simple, sensitive, and specific detection of HeLa cells using Fe 3 O 4 @SiO 2 -Apt to selectively capture and magnetically separate target cancer cells, and using UIO-66(Fe/Zr) NPs to output amplified colorimetric signals. Through designing appropriate metal nodes in the given MOFs, the prepared UIO-66(Fe/Zr) NPs show desirable integrated properties, that is, the intrinsic peroxidase-like activity originated from Fe nodes, and direct binding to the cell surface using the Zr-O-P coordination bond between phosphate units in the phospholipid bilayer of the cell membrane and zirconium nodes. These unique properties ensured high sensing sensitivity and broke through the limitation of the requirement of labeling two antibodies or aptamers. The constructed colorimetric aptasensor could achieve visual detection of HeLa cells in the range of 10 3 -10 4 cells/mL with a detection limit of 481 cells/mL. Such a novel single aptamer-dependent colorimetric sandwich-type biosensor has great potential in the diagnosis and treatment evaluation of cancer. Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Ethical Committee (IEC) of Guangxi Normal University (protocol code 202207-003).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available within the article and its Supplementary Materials. Other data that support the findings of this study are available upon request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.