Prostate-Specific Antigen Monitoring Using Nano Zinc(II) Metal–Organic Framework-Based Optical Biosensor

Background: The prostate-specific antigen (PSA) is an important cancer biomarker that is commonly utilized in the diagnosis of prostate cancer. The development of a PSA determination technique that is rapid, simple, and inexpensive, in addition to highly accurate, sensitive, and selective, remains a formidable obstacle. Methods: In this study, we developed a practical biosensor based on Zn(II) metal–organic framework nanoparticles (Zn-MOFs-NPs). Many spectroscopic and microanalytical tools are used to determine the structure, morphology, and physicochemical properties of the prepared MOF. Results: According to the results, Zn-MOFs-NPs are sensitive to PSA, selective to an extremely greater extent, and stable in terms of chemical composition. Furthermore, the Zn-MOFs-NPs did not exhibit any interferences from other common analytes that might cause interference. The detection limit for PSA was calculated and was 0.145 fg/mL throughout a wide linear concentration range (0.1 fg/mL–20 pg/mL). Conclusions: Zn-MOFs-NPs were successfully used as a growing biosensor for the monitoring and measurement of PSA in biological real samples.


Introduction
In males, the prostate gland is a common site for the development of prostate cancer, one of the most rapidly progressing forms of malignant tumors [1][2][3][4]. It is the second highest cause of cancer patient fatalities in the United States, and constitutes 7.2% of the total number of cancer cases in Egypt, as estimated by Global Cancer Observatory (GLOBOCAN) in December 2020, which makes it the fifth most prevalent cancer [5][6][7]. In the vast majority of prostate cancer cases, symptoms do not manifest until later stages [8]. It is preferable to diagnose this sort of tumor as early as possible to reduce the mortality rate using normal therapy procedures [9]. Recent statistics from the World Health Organization (WHO) indicate an estimated prostate cancer death rate of around 1.7 million per year, with a rise of approximately 0.5 million by 2030 [6,10].
PSA, or prostate-specific antigen, is a glycoprotein made up of 237 amino acids that is released by the prostate gland [11]. It is used as an important biomarker for diagnosing prostate cancer by measuring its concentration in blood samples [11]. Serum PSA concentrations between 4.0 and 10.0 ng/mL are considered normal; a concentration of PSA more than 10.0 ng/mL is indicative of a carcinoma of the prostate in its first stages [12,13].
Owing to the widespread prevalence of malignant neoplasm of the prostate in men, several scientific and medical research investigations have focused on the development

PL-Measurements Procedures
By dissolving an adequate quantity of Zn-MOFs-NP in DMSO, a concentration of (1 mM) stock solution was obtained. A working solution (10 mM) was produced by diluting the stock solution with deionized water. Then, the sample solution was evaluated for PL measurements to determine the optimal excitation wavelength by choosing the maximum wavelength emission. The PL spectrum of Zn-MOFs-NP (10 mM) was investigated against appropriate concentrations that were freshly prepared of PSA, as biomolecules and significant tumor biomarkers. The PL-intensity at ň Em = 366 nm after an excitation wavelength of 305 nm was shown to be linearly related to PSA concentration between 0.1 and 0.2 fg/mL under the most ideal PL measuring circumstances. Parameters of the equation, including the correlation coefficient, were calculated using the least-squares method of statistics. The equation that was used was (Y = n + n'X), where Y represents the PL intensity of the Zn-MOFs-NP at em = 372 nm, n represents the intercept, n' represents the slope, and X is the concentration of PSA. Moreover, the LOQ and LOD were determined using the following equations: [61][62][63]. (S) is the standard-error value of the PL intensity; (b) is the slope of the linear-graph. Zn-MOFs-NP PL spectra were also examined to those of true blood samples that had been treated with various amounts of PSA.

PSA Determination by MOF in Real-Samples
The serum samples that are considered to be representative were taken from a disposable sample that was located in a medical laboratory. The samples were evaluated and processed in accordance with the standard recommendations and safety procedures.

Results
The Zn-MOFs-NPs were made by reacting zinc II acetate with the N1,N3-bis(2-(3-((l1oxidaneyl)carbonyl)-5-aminobenzamido)phenyl)-5-aminoisophthalamide compound in nano form represented as a linker in a simple way, as shown in Figure S1 (Reaction Scheme). After obtaining a precipitate with a somewhat off-white color, the substance was filtered, then washed, and, lastly, dried. Based on the collected micro/analytical data, a suggested structure was performed and evaluated.

FE-SEM, EDX and HR-TEM
The FE-SEM and EDX images of Zn-MOFs-NP are presented in (Figure 1a-c). The FE-SEM images of Zn-MOFs-NP at different magnifications (Figure 1a,b) looked to be an aggregation of inconsistent square forms (1a) and nearly inconsistent square wood shapes with proportionally sized dimensions of around 118 nm (1b). The mapping analysis of the Zn-MOFs-NPs using EDX ( Figure 1c and Table 1) revealed the existence of carbon, oxygen, nitrogen, and zinc as a building block element in every single particle surface. EDX mapping (Figure 1c) confirmed the formation of the Zn-MOFs-NP structure by displaying a uniform distribution of block components throughout the cross-section. Similarly, the mapping element percentages from the EDX Table (Table 1) were in close agreement with the approximate proportion of theoretical elements. Theoretically, C is 40.18, N is 6.83, O is 28.99, and Zn is 18.23. In practice, C is 40.00, N is 6.57, O is 34.87, and Zn is 18.56. The Zn-MOFs-NP TEM picture (Figure 1d) shows irregular square nanosheets with a moderate size of around 120 nm. The SAED and high magnification pictures of the 3D nanostructure of square sheets are shown in (Figure 1e,f). High-resolution TEM (1e) and its selected area electron diffraction (SAED) pattern (1f) suggest the high crystallinity of Zn-MOF, which was confirmed by XRD.

Mass Spectrum
The mass spectrum of the Zn-MOFs-NP compound ( Figure S2) and the proposed fragmentation strategy ( Figure S3) were illustrated. The figures depicted the m/z peaks that were perfectly consistent with the suggested empirical formula (C 48 H 82 N 7 O 26 Zn 4 ), as computed theoretically and validated by C\H\N-analysis, a molecular ion-peak was observed at 1434.730 m/z for the Zn-MOFs-NP compound. Following fragmentation of the ion with m/z = 1434.730, Figure S3 also displays several significant peaks at m/z = 943.0, 716.0, 488.0, 229.0, 171.0, 149.0, 108.0, and 65.0 caused by the loss of (ethanol and water) molecules and the subsequent breakdown of the organic structure. The Zn-MOFs-NP compound fractured in most cases in accordance with the mass spectrum, computed theoretical fragmentations, and presumed structures of molecules.

UV-Vis and FT-IR Spectra
In contrast to the nano organic linker, the spectra chart of the IR spectroscopy of Zn-MOFs-NP is displayed in Figure S4 Figure S5. These bands might be caused by LMCT and intra-ligand -*, n-* [68]. The BGE spectra in Figure S6 further show that the Zn-MOFs-NP has lower BGE values than the linker at 1.79, 2.95, and 3.50 eV due to conjugation inside the N1,N3-bis(2-(3-((l1oxidaneyl)carbonyl)-5-aminobenzamido)phenyl)-5-aminoisophthalamide skeleton, which results in a higher BEG for the HOMO valance [41,55].

XRD and XPS Analysis
The XRD chart of different Zn-MOFs-NP compounds was presented in (Figure 2a). The XRD patterns of the Zn-MOFs-NP showed sharp strong peaks, indicating that the high crystalline-phase of the Zn-MOFs-NP had developed. Moreover, the diffraction patterns corresponded to normal Zn-MOF and other produced Zn-MOFs-NP XRD patterns, demonstrating the effectiveness of the Zn-MOFs-NP Synthesis method [69,70]. The nano crystallite size was estimated by the Scherrer equation provided in Tables S1 and S2, which showed the crystallite size was in the range of 120 nm. This finding has corroborated the SEM and TEM findings.

Thermal Behavior and Stability of the Zn-MΟFs-NP
TGA/DSC plots of Zn-MΟFs-NP indicated that the Zn-MΟFs-NP compound broke down in four phases (Figure 2f). At temperatures ranging from 65 to 226.0 °C, the initial and subsequent weight losses are attributable to the liberation of C2H5ΟH and intra/inter H2Ο molecules. The organic skeleton began to degrade around 440.0 °C. This resulted in the third and fourth breakdown phases. The remainder is zinc (18.11 percent). The discussed data agreed with the results of mass spectrometry and XRD. The thermogravimetric behavior of the Zn-MΟFs-NP further suggests that at temperatures up to 400.0 °C the Zn-MΟFs-NP is thermally stable [72,73].
The whole presented data and discussion may be used to determine the structure of prepared Zn-MΟFs-NP that exists in three dimensions and its exceptional molecular

Thermal Behavior and Stability of the Zn-MOFs-NP
TGA/DSC plots of Zn-MOFs-NP indicated that the Zn-MOFs-NP compound broke down in four phases (Figure 2f). At temperatures ranging from 65 to 226.0 • C, the initial and subsequent weight losses are attributable to the liberation of C 2 H 5 OH and intra/inter H 2 O molecules. The organic skeleton began to degrade around 440.0 • C. This resulted in the third and fourth breakdown phases. The remainder is zinc (18.11 percent). The discussed data agreed with the results of mass spectrometry and XRD. The thermogravimetric behavior of the Zn-MOFs-NP further suggests that at temperatures up to 400.0 • C the Zn-MOFs-NP is thermally stable [72,73].
The whole presented data and discussion may be used to determine the structure of prepared Zn-MOFs-NP that exists in three dimensions and its exceptional molecular surface (Figure 3a,b). Accordingly, the 3D structure of Zn-MOF plates and morphology have intrinsic properties such as photonic bandgap, microporosity, high-spatial density, low surface energy, the defects and edge positions, complexity over length, and scales ranging and integrated with functional low-dimensional building blocks which can enhance the performance of sensing properties. Geometry with special crystal facets can provide a high large surface area and active sites, which is critically significant for highly efficient sensors. surface (Figure 3a,b). Accordingly, the 3D structure of Zn-MOF plates and morphology have intrinsic properties such as photonic bandgap, microporosity, high-spatial density, low surface energy, the defects and edge positions, complexity over length, and scales ranging and integrated with functional low-dimensional building blocks which can enhance the performance of sensing properties. Geometry with special crystal facets can provide a high large surface area and active sites, which is critically significant for highly efficient sensors.

Photoluminescence Investigation
Specifically, the PL curve was made via graphing the data of the excitation spectrum versus the emission spectra of Zn-MΟFs-NP (Figure 4a). Zn-MΟFs-NP photoluminescence (PL) spectra were measured and plotted (at different excitation wavelengths) in Figure S7. At an excitation wavelength of 305 nm (10 mM), the PL spectrum of Zn-MΟFs-NP revealed a rise of emission band located at 366 nm. Furthermore, Zn-MΟFs-NPs may be employed as optical biosensors for PSA detection and measurement.
ZMOF-NPs photoluminescence spectrum was analyzed in relation to a range of prostate-specific antigen concentrations (Figure 4b). It was found that the increase in the PSA concentrations significantly enhanced the Zn-MΟFs-NP PL peak intensity in a PSA range of concentrations (0.1-0.2 fg/mL) with a small red-shift (about six nm) from 366.0 to 372.0 nm. After taking into consideration technique evaluations and statistical characteristics, the results demonstrated that ZMOF-NPs has the potential to be deemed a suitable biosensor based on spectrofluorimetric phenomena for the detection and quantification of PSA.

Photoluminescence Investigation
Specifically, the PL curve was made via graphing the data of the excitation spectrum versus the emission spectra of Zn-MOFs-NP (Figure 4a). Zn-MOFs-NP photoluminescence (PL) spectra were measured and plotted (at different excitation wavelengths) in Figure S7. At an excitation wavelength of 305 nm (10 mM), the PL spectrum of Zn-MOFs-NP revealed a rise of emission band located at 366 nm. Furthermore, Zn-MOFs-NPs may be employed as optical biosensors for PSA detection and measurement.

Method Validation
The calibration curve demonstrates the relationship between the PL intensi ZMOFNP measured at ƛEm = 372.0 nm and the concentration of PSA in the sample, w may give rise in between 0.1 and 0.2 fg/mL ( Figure S8). The linear-dynamic relation for the Zn-MΟFs-NP PL biosensor was observed (Figure 4c) under optimal circumsta ZMOF-NPs photoluminescence spectrum was analyzed in relation to a range of prostate-specific antigen concentrations (Figure 4b). It was found that the increase in the PSA concentrations significantly enhanced the Zn-MOFs-NP PL peak intensity in a PSA range of concentrations (0.1-0.2 fg/mL) with a small red-shift (about six nm) from 366.0 to 372.0 nm. After taking into consideration technique evaluations and statistical characteristics, the results demonstrated that ZMOF-NPs has the potential to be deemed a suitable biosensor based on spectrofluorimetric phenomena for the detection and quantification of PSA.

Method Validation
The calibration curve demonstrates the relationship between the PL intensity of ZMOFNP measured at ň Em = 372.0 nm and the concentration of PSA in the sample, which may give rise in between 0.1 and 0.2 fg/mL ( Figure S8). The linear-dynamic relationship for the Zn-MOFs-NP PL biosensor was observed (Figure 4c) under optimal circumstances for PL measurements. Rising PSA concentration significantly increased the spectrum PL intensities (Figure 4c). Over the range of interest (0.1-1000.0 fg/mL), the calibration curve was graphed linearly.
Zn-MOFs-NP PL-intensity = 105.21 log [PSA] + 523.01 with r 2 = 0.983. The limit of detection and quantification (LOD and LOQ) of the optical photoluminescence biosensor based on ZN-MOFS-NP was calculated to be 0.145 and 0.438 fg/mL, respectively, where Table S3 summarizes the results of the analysis of data using PL regression. The suggested optical photoluminescence biosensor's low LOD and LOQ values and large linear ranges of concentrations are validated by table data. Furthermore, (Table 2) compares the new biosensor's performance to other prior studies for PSA measurement and determination, where the present optical photoluminescence biosensor has a lower LOQ and LOD, and a broader linear detection PSA range compared to previous methods of detection. Studies were conducted to determine the selectivity and specificity of the current optical photoluminescence Zn-MOFs-NP-based biosensor in the presence of competing analytes, such as biomolecules and other tumor biomarkers. Through the use of the PL spectrum of Zn-MOFs-NP (10.0mM) against PSA (100.0 fg/mL), CEA "carcinoembryonic antigen" (10.0 ng/mL), AFP "alpha-fetoprotein" (10.0 ng/mL), and CK-T "creatine kinase total" (10.0 ng/mL), selectivity and specificity studies were collated and exhibited in a histogram (Figure 4d). From the histogram, it can be noted that the luminescence intensity in cases of PSA is highly enhanced, whereas the other interfering analytes (similar biomarkers or organic molecules) do not make significant changes in the luminescence intensity of the Zn-MOFs-NP-based biosensor, which demonstrates the high selectivity and specificity of the proposed biosensor.
The curve demonstrates that PSA induced a considerable rise in the intensity of the Zn-MOFs-NP PL with a minor redshift, but the other interfering matrix had no reaction. As a result, we may infer that Zn-MOFs-NPs are very specific and selective for PSA when compared to other interfering matrices. Zn-MOFs-NP-based optical photoluminescence biosensors for PSA detection and measurements in genuine human blood serum samples were evaluated for their precision, accuracy, recovery, and usability. In this study, the PSA standard was spiked into blood samples at doses of 1.0, 100.0, 500.0, 1000.0, and 2000.0 fg/mL.
Three separate repetitions of each test were performed ( Table 3). The mathematical calculations and statistical evaluations showed that the mean values (X) were suitable and in the range. The suggested method is precise and accurate since the observed sharp decrease in the values of relative error (RE%) averaged (1.01%) and the relative standard deviation (SD) averaged 4.270%. Importantly, the average percent recovery (RC%) was 99.02%, proving the existing optical photoluminescence biosensor is sensitive enough to detect prostate-specific antigen as an important cancer biomarker diagnostic for the prostate with pinpoint precision at very low concentrations.

Interaction Mechanism
The fluorescence mechanism of the enhancement process may be extrapolated from (Figure 4b), which indicates that when PSA concentrations increased, so did the PL-emission intensities of Zn-MOFs-NP. The shift in Zn-MOFs-NP emission intensities recorded by increasing PSA concentrations indicates the nature of the MOF-PSA interaction. The observed biochemical sensing behavior of Zn-MOFs-NP towards PSA might be ascribed to PSA's remarkable affinity for the amine group lone pair of electrons inside Zn-MOFs-NP [64]. Furthermore, because the Zn-MOFs-NPs have low-lying "*orbitals" in amine groups of phenyl rings, the fluorescent performance of the Zn-MOFs-NP and further explanation that the interaction mechanism might be because of molecular-orbital transitions inside ligandmetal charge transfer (LMCT) resulted from intra-ligand n/-* transitions. Furthermore, the increased probability of Zn-MOFs-NP chelating with PSA as a biological target may be explained by the potential of "electron" delocalization inside the chelation ring and partial sharing of opposing positive charges with the donor-groups. According to chelation theory, this delocalization and charge-sharing process increases the lipophilic properties of the Zn-MOFs-NP and, as a result, decreases the polarity of the zinc-ion [52], implying that the interaction of Zn-MOFs-NP with PSA may be due to the formation of covalent bonds [65].

Conclusions
This scientific study describes an optical biosensor that employs photoluminescence and is based on a novel, promising Zn-MOFs-NP that has been thoroughly evaluated. The Zn-MOFs-NP morphology looked to be inconsistent square wood forms with an appropriate size of around 118 nm. The thermal stability measurements further demonstrated that the Zn-MOFs-NPs were considered stable. The photoluminescence investigation findings suggested that Zn-MOFs-NP might be employed as an advantageous optical biosensor for determining PSA content in actual human blood serum. From a statistical perspective, the current method served its intended function well. The provided results of the suggested approach showed lower PSA LOD with a response that was faster, easier, and cheaper than those reported in the literature. Moreover, the data unveiled in the not-so-distant future will be an essential piece for monitoring and quantifying PSA, a useful diagnostic tool for identifying prostate cancer early on-which is the most frequent malignancy in males worldwide-and therefore aids in monitoring public concerns about the health of men.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/bios12110931/s1, Figure S1: The proposed mechanism of the Zn-MOFs-NP synthesis; Figure S2: Mass spectrum of the Zn-MOFs-NP; Figure S3: The proposed fragmentation Scheme of the Zn-MOFs-NP; Figure S4: FT-IR spectra of Zn-MOFs-NP and nano organic linker; Figure S5: The electronic-reflection spectra of Zn-MOFs-NP and nano organic linker; Figure S6: The bandgap energy spectra of Zn-MOFs-NP and nano organic linker; Figure S7: The PL emission spectra at different excitation wavelength for the Zn-MOFs-NP; Figure S8: A full relationship (calibration graph) between the PL intensity of Zn-MOFs-NP and the logarithm PSA concentration (log [PSA]); Table S1: Summary of XRD data, Miller indices and interplanar distances of Zn-MOFs-NP; Table S2: Summary of calculated crystallite size of Zn-MOFs-NP at different position on XRD patterns; Table S3: Sensitivity and regression parameters for Zn-MOFs-NP chemosensor.

Informed Consent Statement:
In this study, we did not use the samples in any research involving human participants or research involving physical interventions on study participants or involving processing of personal data but conducted the research according to the method described in the article. Data Availability Statement: All data generated or analyzed during this study are included in this published article (and its supplementary information files).