Stable DNA Aptamer–Metal–Organic Framework as Horseradish Peroxidase Mimic for Ultra-Sensitive Detection of Carcinoembryonic Antigen in Serum

Carcinoembryonic antigen (CEA) is an important broad-spectrum tumor marker. For CEA detection, a novel type of metal–organic framework (MOF) was prepared by grafting CEA aptamer-incorporated DNA tetrahedral (TDN) nanostructures into PCN-222 (Fe)-based MOF (referred as CEAapt-TDN-MOF colloid nanorods). The synthesized CEAapt-TDN-MOF is a very stable detection system due to the vertex phosphorylated TDN structure at the interface, possessing a one-year shelf-life. Moreover, it exhibits a significant horseradish peroxidase mimicking activity due to the iron porphyrin ring, which leads to a colorimetric reaction upon binding toward antibody-captured CEA. Using this method, we successfully achieved the highly specific and ultra-sensitive detection of CEA with a limit of detection as low as 3.3 pg/mL. In addition, this method can detect and analyze the target proteins in clinical serum samples, effectively identify the difference between normal individuals and patients with colon cancer, and provide a new method for the clinical diagnosis of tumors, demonstrating a great application potential.


Introduction
The metal-organic framework (MOF) has been widely used in the fields of catalysis, separation, and sensing due to its unique crystalline porosity, flexible tailorability, and large specific surface area. Post-synthetic modification of MOF is mainly used to adjust the physical and chemical properties of the porous framework structure, so as to endow the MOF with specific functions for a variety of application fields [1,2]. Especially in the biosensor field, the development of biomolecule-functionalized MOF-based colloid nanoparticles is a research hotspot [3,4]. Biomolecule functionalization can effectively improve the stability and biocompatibility of MOF, resulting in significant performance even in a complex biological environment [5][6][7]. In addition, a large number of biomolecules can be designed based on the specific molecular recognition principle, such as aptamer technology [8] and peptide phage display technology [9], which can endow MOFs with specific recognition ability, thus leading to enhanced sensing performance. In principle, owing to the incorporated large amounts of organic ligands and the large specific surface area, it is easier to achieve functionalized modifications of MOF by biomolecules with Scheme 1. (A) Schematic diagram of the preparation process of CEAapt-TDN-MOF. First, Zirconia dichloride octahydrate and meso-tetra (4-carboxyphenyl) porphine ferric chloride (Fe-TCPP) were mixed to fabricate PCN-222 (Fe)-based metal-organic framework (MOF). Then, the CEAapt-TDN-MOF colloid nanorods were prepared by grafting carcinoembryonic antigen (CEA) aptamer-incorporated DNA tetrahedral (TDN) nanostructures. (B) Schematic diagram of the principle of constructing a colorimetric immunosensor for detecting CEA based on the CEAapt-TDN-MOF, where 3,3′,5,5′-tetramethylbenzidine (TMB) was used as the substrate.

Preparation and Characterization of CEAapt-TDN-MOF Colloid Nanorods
As shown in Scheme 1, a nano-sized MOF material PCN-222 (Fe) was synthesized by using tetravalent zirconium ion and porphyrin ring Fe-TCPP as the precursor material. This PCN-222 (Fe) could exhibit very good structural stability, excellent biocompatibility, and horseradish peroxidase-like activity due to the presence of an iron porphyrin ring. In addition, a large number of unsaturated zirconium metal sites on the surface of the material might facilitate the post modification [28]. Particularly, the four single strands of the TDN structure were designed and synthesized (Table 1), in which the 5′ ends of the three strands were phosphorylated, and a piece of recognition probe sequence (i.e., the nucleic acid aptamer sequence of CEA) was added to the 5′ end of the fourth strand. Through the self-assembly behavior of DNA based on the Watson-Crick complementary pairing principle, a TDN structure assembled by three phosphorylated vertices was obtained. Reusing the strong interaction of phosphate groups with zirconium ions, the obtained TDN was easily grafted onto the surface of PCN-222 (Fe), which constitutes the final product CEAapt-TDN-MOF colloid nanorods. Since the TDN is a rigid structure, a good assembly layer for molecular recognition was formed at the interface of MOFs, which might facilitate the detection of targets. Since the aptamer sequence for CEA recognition was incorporated in the TDN structure, the constructed CEAapt-TDN-MOF was potentially a high-performance biosensor. Owing to the iron porphyrin ring, it might exhibit catalytic oxidation activity by using TMB as the chromogenic substrate for the detection of CEA. Scheme 1. (A) Schematic diagram of the preparation process of CEA apt -TDN-MOF. First, Zirconia dichloride octahydrate and meso-tetra (4-carboxyphenyl) porphine ferric chloride (Fe-TCPP) were mixed to fabricate PCN-222 (Fe)-based metal-organic framework (MOF). Then, the CEA apt -TDN-MOF colloid nanorods were prepared by grafting carcinoembryonic antigen (CEA) aptamerincorporated DNA tetrahedral (TDN) nanostructures. (B) Schematic diagram of the principle of constructing a colorimetric immunosensor for detecting CEA based on the CEA apt -TDN-MOF, where 3,3 ,5,5 -tetramethylbenzidine (TMB) was used as the substrate.

Preparation and Characterization of CEA apt -TDN-MOF Colloid Nanorods
As shown in Scheme 1, a nano-sized MOF material PCN-222 (Fe) was synthesized by using tetravalent zirconium ion and porphyrin ring Fe-TCPP as the precursor material. This PCN-222 (Fe) could exhibit very good structural stability, excellent biocompatibility, and horseradish peroxidase-like activity due to the presence of an iron porphyrin ring. In addition, a large number of unsaturated zirconium metal sites on the surface of the material might facilitate the post modification [28]. Particularly, the four single strands of the TDN structure were designed and synthesized (Table 1), in which the 5 ends of the three strands were phosphorylated, and a piece of recognition probe sequence (i.e., the nucleic acid aptamer sequence of CEA) was added to the 5 end of the fourth strand. Through the selfassembly behavior of DNA based on the Watson-Crick complementary pairing principle, a TDN structure assembled by three phosphorylated vertices was obtained. Reusing the strong interaction of phosphate groups with zirconium ions, the obtained TDN was easily grafted onto the surface of PCN-222 (Fe), which constitutes the final product CEA apt -TDN-MOF colloid nanorods. Since the TDN is a rigid structure, a good assembly layer for molecular recognition was formed at the interface of MOFs, which might facilitate the detection of targets. Since the aptamer sequence for CEA recognition was incorporated in the TDN structure, the constructed CEA apt -TDN-MOF was potentially a high-performance biosensor. Owing to the iron porphyrin ring, it might exhibit catalytic oxidation activity by using TMB as the chromogenic substrate for the detection of CEA. First of all, the scanning electron microscope (SEM) was used to observe the structure and morphology of the MOF material PCN-222 (Fe), as shown in Figure 1A. Unlike the previously synthesized sphere MOF [29], this time, the obtained MOF material had a rodlike structure with a length of 400-600 nm and a width of about 100 nm. To further verify the ingredients of PCN-222 (Fe), the samples were analyzed by powder X-ray diffraction (PXRD, Figure 1B). The results show that the PXRD spectra of the PCN-222 (Fe) had sharp characteristic diffraction peaks and were highly similar to the simulation results of PCN-222 (Fe) single crystals, indicating that the PCN-222 (Fe)-based MOF material was successfully synthesized. In addition, taking advantage of the DNA self-assembly effect, the A, B, and C single-strand DNA and D chains with phosphorylation modification were annealed to form a TDN structure. In order to study the TDN structure, F, G, and H single-strand DNA (which were similar sequences without phosphorylation modification, with respect to A, B, C single-strand DNA) and D chains were used for agarose gel electrophoresis ( Figure 1C). The first lane is a DNA marker band, and the other lanes are a single-stranded DNA of D, a DNA assembled by two chains of D, a DNA assembled by three chains of D/F/G, and a TDN structure assembled by four chains of D/F/G/H. With the increasing of strands, the electrophoresis rate of the nucleic acid structure decreased gradually, indicating that a TDN structure was successfully obtained. Then, using the strong interaction between phosphate and zirconium ions, PCN-222 (Fe) was incubated with phosphorylated TDN to prepare CEA apt -TDN-MOF. In Figure 1D, the charge change before and after TDN modification was clearly observed by zeta potential analysis. There was a positive charge on the surface of PCN-222 (Fe) at the beginning, and the zeta potential was +18.6 mV. After the TDN functionalization, it shows that the charge changes to negative value of −31.4 mV, indicating that TDN might be successfully grafted on the surface of PCN-222 (Fe). In order to further explore the successful modification of TDN on the MOF nanorods, the D chain was replaced by an E chain with a large number of T base sequences at the end, and we assembled a new tetrahedral structure of TDN' with four single strands of A, B, C, and E DNA. At the same time, the I chain with a multi-A base sequence was grafted on gold nanoparticles with a size of about 13 nm to prepare DNA-AuNPs. Using the complementary pairing of the sequence of A-T bases, a strong molecular recognition event could occur between DNA-AuNPs and the newly prepared TDN'-MOFs. As shown in Figure 1E, the interaction between the two was directly observed by transmission electron microscope (TEM). A large number of AuNPs surround the surface of TDN'-MOFs, indicating a strong molecular recognition. These results fully demonstrate that a DNA tetrahedron was successfully grafted on the surface of PCN-222 (Fe), that is, the CEAapt-TDN-MOF was successfully prepared. Moreover, the prepared CEAapt-TDN-MOF might efficiently detect its target molecule.

Horseradish Peroxidase Mimic Activity Study
To achieve the application purpose of serum tests, the horseradish peroxidase mimic activity study was designed to be carried out in 1 × PBS containing 0.05% Tween-20 (PBST, pH ~7), where the CEAapt-TDN-MOF remained 27% of the maximum activity ( Figure S2). To investigate the horseradish peroxidase mimicking activity of our CEAapt-TDN-MOF, H2O2 was added to the experimental system to catalyze the oxidation of TMB substrate, which resulted in the color change of the solution. Figure 2A shows the UV absorption In order to further explore the successful modification of TDN on the MOF nanorods, the D chain was replaced by an E chain with a large number of T base sequences at the end, and we assembled a new tetrahedral structure of TDN' with four single strands of A, B, C, and E DNA. At the same time, the I chain with a multi-A base sequence was grafted on gold nanoparticles with a size of about 13 nm to prepare DNA-AuNPs. Using the complementary pairing of the sequence of A-T bases, a strong molecular recognition event could occur between DNA-AuNPs and the newly prepared TDN'-MOFs. As shown in Figure 1E, the interaction between the two was directly observed by transmission electron microscope (TEM). A large number of AuNPs surround the surface of TDN'-MOFs, indicating a strong molecular recognition. These results fully demonstrate that a DNA tetrahedron was successfully grafted on the surface of PCN-222 (Fe), that is, the CEA apt -TDN-MOF was successfully prepared. Moreover, the prepared CEA apt -TDN-MOF might efficiently detect its target molecule.

Horseradish Peroxidase Mimic Activity Study
To achieve the application purpose of serum tests, the horseradish peroxidase mimic activity study was designed to be carried out in 1 × PBS containing 0.05% Tween-20 (PBST, pH~7), where the CEA apt -TDN-MOF remained 27% of the maximum activity ( Figure S2). To investigate the horseradish peroxidase mimicking activity of our CEA apt -TDN-MOF, H 2 O 2 was added to the experimental system to catalyze the oxidation of TMB substrate, which resulted in the color change of the solution. Figure  To intuitively compare the differences between each group, the UV absorption spectra of TMB in each group's solution was recorded at a wavelength range of 550-700 nm (with a peak at 652 nm), and the results remained in accordance with the color reaction changes. The group a and group b spectra were similar, showing no UV absorption peak at this wavelength range. Meanwhile, both group c and group d had very high peaks, indicating the high completion of catalytic reaction. As illustrated in Figure 2C, the TMB oxidation reaction was catalyzed by the CEA apt -TDN-MOF via Fenton reaction: Fe 2+ + H 2 O 2 → Fe 3+ + HO·+ OH − . Moreover, group d had a slightly lower peak than group c but almost negligible, indicating that TDN functionalization had very little effect on the catalytic activity of PCN-222 (Fe), which could effectively guarantee the feasibility of the experiment. In addition, the catalytic ability of CEA apt -TDN-MOF was studied by recording the time-varying absorbance curve of TMB at 652 nm. As shown in Figure 2B, the kinetic curve showed that the catalytic ability of CEA apt -TDN-MOF increased with the increase of concentration, and the corresponding concentrations of each curve were 0, 6.25, 12.5, 25, 35, and 50 µg/mL (a to f), respectively. Therefore, the catalytic performance of the system could be achieved by adjusting the concentration of CEA apt -TDN-MOF in practical applications. Moreover, it was found that this CEA apt -TDN-MOF exhibited superior catalytic effect compared with nature horseradish peroxidase ( Figure S3), indicating the great potential of this detection system. To intuitively compare the differences between each group, the UV absorption spectra of TMB in each group's solution was recorded at a wavelength range of 550-700 nm (with a peak at 652 nm), and the results remained in accordance with the color reaction changes. The group a and group b spectra were similar, showing no UV absorption peak at this wavelength range. Meanwhile, both group c and group d had very high peaks, indicating the high completion of catalytic reaction. As illustrated in Figure 2C, the TMB oxidation reaction was catalyzed by the CEAapt-TDN-MOF via Fenton reaction: Fe 2+ + H2O2 → Fe 3+ + HO·+ OH − . Moreover, group d had a slightly lower peak than group c but almost negligible, indicating that TDN functionalization had very little effect on the catalytic activity of PCN-222 (Fe), which could effectively guarantee the feasibility of the experiment. In addition, the catalytic ability of CEAapt-TDN-MOF was studied by recording the time-varying absorbance curve of TMB at 652 nm. As shown in Figure 2B, the kinetic curve showed that the catalytic ability of CEAapt-TDN-MOF increased with the increase of concentration, and the corresponding concentrations of each curve were 0, 6.25, 12.5, 25, 35, and 50 μg/mL (a to f), respectively. Therefore, the catalytic performance of the system could be achieved by adjusting the concentration of CEAapt-TDN-MOF in practical applications. Moreover, it was found that this CEAapt-TDN-MOF exhibited superior catalytic effect compared with nature horseradish peroxidase ( Figure S3), indicating the great potential of this detection system.

Optimization of Experimental Conditions
First, phosphorylated single-strand DNA (chain J) containing a CEA aptamer was grafted on the surface of PCN-222 (Fe) to prepare CEAapt-MOF for the colorimetric sensing of CEA. The results show that the signal response value of CEAapt-TDN-MOF was higher

Optimization of Experimental Conditions
First, phosphorylated single-strand DNA (chain J) containing a CEA aptamer was grafted on the surface of PCN-222 (Fe) to prepare CEA apt -MOF for the colorimetric sensing of CEA. The results show that the signal response value of CEA apt -TDN-MOF was higher than that of CEA apt -MOF under the same conditions (CEA concentration is 20 ng/mL), which was probably because the rigid structure of TDN could effectively promote the Gels 2021, 7, 181 7 of 13 molecular recognition between the aptamer sequence and target protein, while the flexible single-strand structure might adhere to the surface of the MOF material and reduce the binding efficiency by 35% [30]. Moreover, the detection performance had no change during one year in PBS at 37 • C, indicating the excellent stability of CEA apt -TDN-MOF with respect to CEA apt -MOF (showing an obvious downward trend in detection ability from the sixth month) ( Figure S1). Meanwhile, the shelf-life of the DNA oligo in PBS at 37 • C is only 6 weeks [31]. Therefore, the use of rigid TDN is very necessary for establishing a stable detection system. Then, the CEA aptamer was integrated into DNA tetrahedron so as to construct a colorimetric biosensor based on the molecular recognition ability and oxidation reaction catalytic property. In order to obtain good detection performance, the experimental conditions were optimized according to the response results of the color signal. First of all, the concentration of CEA apt -TDN-MOF was optimized, since the concentration of the material directly determined the catalytic performance and the detection effect. As shown in Figure 3A, when the concentration of CEA apt -TDN-MOF increased continuously, the colorimetric signal increased all the time and tended to be stable when the concentration reached 25 µg/mL, so this concentration was selected for detection use. Then, the concentrations of chromogenic substrate TMB and oxidant H 2 O 2 were optimized, respectively. As shown in Figure 3B,C, the detection effect was optimal when the concentrations of TMB and H 2 O were 2 mM and 0.2 M, respectively. Furthermore, according to the results of Figure 3D, the detection time should be 20 min. Under the optimized conditions, CEA detection was carried out.
Gels 2021, 7, x FOR PEER REVIEW 7 of 13 than that of CEAapt-MOF under the same conditions (CEA concentration is 20 ng/mL), which was probably because the rigid structure of TDN could effectively promote the molecular recognition between the aptamer sequence and target protein, while the flexible single-strand structure might adhere to the surface of the MOF material and reduce the binding efficiency by 35% [30]. Moreover, the detection performance had no change during one year in PBS at 37 °C, indicating the excellent stability of CEAapt-TDN-MOF with respect to CEAapt-MOF (showing an obvious downward trend in detection ability from the sixth month) ( Figure S1). Meanwhile, the shelf-life of the DNA oligo in PBS at 37 °C is only 6 weeks [31]. Therefore, the use of rigid TDN is very necessary for establishing a stable detection system. Then, the CEA aptamer was integrated into DNA tetrahedron so as to construct a colorimetric biosensor based on the molecular recognition ability and oxidation reaction catalytic property. In order to obtain good detection performance, the experimental conditions were optimized according to the response results of the color signal. First of all, the concentration of CEAapt-TDN-MOF was optimized, since the concentration of the material directly determined the catalytic performance and the detection effect. As shown in Figure 3A, when the concentration of CEAapt-TDN-MOF increased continuously, the colorimetric signal increased all the time and tended to be stable when the concentration reached 25 μg/mL, so this concentration was selected for detection use. Then, the concentrations of chromogenic substrate TMB and oxidant H2O2 were optimized, respectively. As shown in Figure 3B,C, the detection effect was optimal when the concentrations of TMB and H2O were 2 mM and 0.2 M, respectively. Furthermore, according to the results of Figure 3D, the detection time should be 20 min. Under the optimized conditions, CEA detection was carried out.

Assay Performance Analysis
After optimization of the conditions, the designed colorimetric sensing method was applied to detect different concentrations of CEA, and the results after stopping the reaction with 1 M sulfuric acid are shown in Figure 4A. The color response gradually became deeper with increasing concentrations of CEA (the concentrations were 0, 0.005, 0.01, 0.02, 0.1, 1.5, 25, and 50 ng/mL, respectively). At the same time, the corresponding UV absorption spectra were also measured, and the results are shown in Figure 4B. By plotting the UV absorption value of each group at 450 nm versus the logarithm of CEA Gels 2021, 7, 181 8 of 13 concentration, it was found that the CEA apt -TDN-MOF exhibited a good linear detection range of 0.01-25 ng/mL, and the detection limit was 3.3 pg/mL (calculated according to the equation: D = 3σ/k, where σ is the relative standard deviation of the blank sample, k is the slope of the calibration line, Figure 4C) [32], showing great potential for CEA detection in human serum. In order to further study the specificity of the sensing system, different proteins such as thrombin, bovine serum albumin (BSA), and alpha-fetoprotein (AFP) were used as controls. It was found that the CEA apt -TDN-MOF exhibited a negligible detection signal toward control proteins (i.e., thrombin, BSA, AFP) even at the concentration of 100 ng/mL. These results demonstrate that our colorimetric sensing system has very high sensitivity and specificity ( Figure 4D).

Assay Performance Analysis
After optimization of the conditions, the designed colorimetric sensing method was applied to detect different concentrations of CEA, and the results after stopping the reaction with 1 M sulfuric acid are shown in Figure 4A. The color response gradually became deeper with increasing concentrations of CEA (the concentrations were 0, 0.005, 0.01, 0.02, 0.1, 1.5, 25, and 50 ng/mL, respectively). At the same time, the corresponding UV absorption spectra were also measured, and the results are shown in Figure 4B. By plotting the UV absorption value of each group at 450 nm versus the logarithm of CEA concentration, it was found that the CEAapt-TDN-MOF exhibited a good linear detection range of 0.01-25 ng/mL, and the detection limit was 3.3 pg/mL (calculated according to the equation: D = 3σ/k, where σ is the relative standard deviation of the blank sample, k is the slope of the calibration line, Figure 4C) [32], showing great potential for CEA detection in human serum. In order to further study the specificity of the sensing system, different proteins such as thrombin, bovine serum albumin (BSA), and alpha-fetoprotein (AFP) were used as controls. It was found that the CEAapt-TDN-MOF exhibited a negligible detection signal toward control proteins (i.e., thrombin, BSA, AFP) even at the concentration of 100 ng/mL. These results demonstrate that our colorimetric sensing system has very high sensitivity and specificity ( Figure 4D). To demonstrate the potential of the CEAapt-TDN-MOF for clinical uses, the detection of CEA in human serum was carried out by using CEAapt-TDN-MOF and confirmed by standardized ELISA. According to clinical studies, the level of CEA in serum is closely related to the occurrence of colon cancer [33,34]. Therefore, the serum samples from healthy individuals, patients with early colon cancer, and patients with advanced colon cancer were used. As shown in Figure 5, the concentration of CEA in serum of patients was significantly higher than that of healthy individuals, and there was also a significant difference in the level of CEA between patients with early and late-stage colon cancer. On the whole, with the improvement of the malignant degree of colon cancer, the content of CEA in blood also increased, indicating that our method has a broad application prospect in tumor diagnosis. To demonstrate the potential of the CEA apt -TDN-MOF for clinical uses, the detection of CEA in human serum was carried out by using CEA apt -TDN-MOF and confirmed by standardized ELISA. According to clinical studies, the level of CEA in serum is closely related to the occurrence of colon cancer [33,34]. Therefore, the serum samples from healthy individuals, patients with early colon cancer, and patients with advanced colon cancer were used. As shown in Figure 5, the concentration of CEA in serum of patients was significantly higher than that of healthy individuals, and there was also a significant difference in the level of CEA between patients with early and late-stage colon cancer. On the whole, with the improvement of the malignant degree of colon cancer, the content of CEA in blood also increased, indicating that our method has a broad application prospect in tumor diagnosis.

Conclusions
In this work, we successfully functionalized DNA tetrahedral nanostructures onto PCN-222 (Fe) materials with simulated horseradish peroxidase activity and constructed a colorimetric immune biosensor for the ultra-sensitive detection of CEA. The phosphorylated DNA tetrahedral structure could be effectively fixed on the PCN-222 (Fe) surface through strong coordination with zirconium ions, thus bringing very stable performance.

Conclusions
In this work, we successfully functionalized DNA tetrahedral nanostructures onto PCN-222 (Fe) materials with simulated horseradish peroxidase activity and constructed a colorimetric immune biosensor for the ultra-sensitive detection of CEA. The phosphorylated DNA tetrahedral structure could be effectively fixed on the PCN-222 (Fe) surface through strong coordination with zirconium ions, thus bringing very stable performance. Moreover, the rigid structure of TDN greatly promoted the binding of the aptamer sequence at the end of the tetrahedron to the target. As a consequence, the strong molecular recognition ability and high enzyme catalytic activity ensure that the colorimetric sensing method has very high specificity and sensitivity, and the detection limit is as low as 3.3 pg/mL (superior with respect to other sensors of the same type, Table S1) [3,4,[35][36][37][38][39][40][41], even in clinical serum samples, so it has great application potential. In addition, the strategy proposed in this work can be extended to other aptamer sequences, so it has high universality.

Synthesis of Metal-Organic Framework PCN-222 (Fe)
First, 38 mg of zirconia dichloride octahydrate was dissolved with 6.5 mg of Fe-TCPP in 16.3 mL of DMF and sonicated for 1 min to mix well. Then, 0.25 mL of dichloroacetic acid was added into the mixed solution, transferred into a Teflon-lined hydrothermal reaction tank, and tightened to sit in a 180 • C oven for 18 h to heat. After the reaction, the as-synthesized nanocrystals were obtained by the method of centrifugation (10,500 rpm, 10 min), which was immediately followed by centrifugal washing with DMF and ethanol solution to remove the precursor species that did not participate in the reaction, and repeated 3 times. Finally, the solid was dried under vacuum at 80 • C overnight for further use.

Preparation and Characterization of TDN Structures
The preparation process of TDN with aptamer sequences was described as following: four single-stranded DNAs (A, B, C, D) were mixed in TM buffer (10 mm Tris HCl, 50 mM MgCl 2 , pH 8.0) at equal ratios. Then, the solution was heated to 95 • C and re-cooled to 4 • C to complete the renaturation process. In addition, the preparation of TDN-bearing poly-T sequences (four single-stranded DNAs, A, B, C, E) was similar to the procedure described above. Finally, the as-prepared two tetrahedron structures were stored at 4 • C for further use.
Agarose gel electrophoresis: the buffer conditions were 1 × TBE, 3% agarose gel, electrophoresis at 100 V for 40 min, and the TDN (four single-stranded DNA of D, F, G, H) concentration was 1 µM.

Preparation of TDN-Functionalized Modified PCN-222 (Fe): CEA apt -TDN-MOF Colloid Nanorods
First, 0.2 mL solution containing TDN (10 µM) was added to 2 mg (783 nmol) PCN-222 (Fe) nanorods and incubated on a shaker for 4 h. During this period, the NaCl solution was added slowly to reduce the electrostatic repulsion, and the final concentration was 0.5 M. Then, the excess nucleic acid structure was removed by centrifugal washing (10,000 rpm, 15 min), and the operation was repeated three times. Finally, it was re-dispersed into TM buffer and stored at 4 • C for use.

Preparation of DNA-AuNPs and TDN-MOFs
First, 1 mL of 40 nm diameter gold nanoparticles was taken from the mother liquor into a low-adsorption EP tube, centrifuged for 15 min (16,200× g) at 4 • C, and resuspended in 1 mL of 10 nM phosphate (PB) buffer. In addition, 100 µM of thiol-modified DNA sequence (strand J) was activated by incubation with 10 nM of TCEP solution for 1 h. Then, 35 µL of activated DNA was added to the above gold nanoparticles solution (the final concentration was 3 µM) and incubated for 16 h. Afterwards, 9 µL of NaCl solution (2 M) was gradually added to the reaction system, and the final salt concentration was 0.1 M, during which we were gently tapping the wall to avoid aggregation. The DNA-modified gold nanoparticles were centrifuged at 4 • C for 15 min (16,600× g) to remove the supernatant and resuscitated with 10 mM PB buffer. Then, 500 nmol PCN-222 (Fe) nanorods decorated with TDN with poly-T sequences were incubated with DNA-modified gold nanoparticles for 2 h to complete the assembly behavior, and finally, DNA-modified gold nanoparticles without the assembly behavior occurring were removed by centrifugation.

Construction of an Immunosensor for the Detection of CEA
First, 100 µL of capture antibody anti-CEA solution (3 µg/mL) was added to each well of a 96-microwell plate and incubated at 4 • C overnight. After the antibody solution was removed, the antibody solution was washed with PBST buffer. Then, 150 µL BSA sealing solution was added to each well. After incubation at 37 • C for 1 h, it was washed 3 times with PBST buffer. Then, each well was incubated with different concentrations of the target protein (CEA) 37 • C for 1 h, the well with only PBST solution was set as the blank control group, and the washing step was repeated three more times. This was immediately followed by the addition of 100 µL of TDN-functionalized modified PCN-222 (Fe) nanorods (25 µg/mL, containing 5 nM PCN-222 (Fe)) to each well, and the wells were sealed with a sealing film and incubated at room temperature for 1 h. Finally, 100 µL of TMB substrate solution (2 mM TMB and 0.2 M H 2 O 2 ) was added with gentle shaking for 20 min, after which 50 µL of stop solution (1 M concentrated sulfuric acid) was added to stop the reaction, and the absorbance intensity was measured at 450 nm with a microplate reader. The standardized ELISA test was performed by following the instructions for the product: EHCEA (96 tests) at thermofisher.com, accessed on 18 October 2021.
Human serum samples from colon cancer patients were obtained from the Second Affiliated Hospital of Nanjing, Southeast University. Informed consent was obtained from