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pH-Regulated Optical Performances in Organic/Inorganic Hybrid: A Dual-Mode Sensor Array for Pattern Recognition-Based Biosensing Qing Yan, Xu-Yin Ding, Zi-Han Chen, Shi-Fan Xue, XinYue Han, Zi-Yang Lin, Miao Yang, Guoyue Shi, and Min Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02603 • Publication Date (Web): 13 Aug 2018 Downloaded from http://pubs.acs.org on August 13, 2018

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Analytical Chemistry

pH-Regulated Optical Performances in Organic/Inorganic Hybrid: A Dual-Mode Sensor Array for Pattern Recognition-Based Biosensing Qing Yan, Xu-Yin Ding, Zi-Han Chen, Shi-Fan Xue, Xin-Yue Han, Zi-Yang Lin, Miao Yang, Guoyue Shi, Min Zhang* School of Chemistry and Molecular Engineering, Shanghai Key Laboratory for Urban Ecological Processes and Eco-Restoration, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China. Email: [email protected] ABSTRACT: Dual-mode optical assays are becoming more popular and attractive, since they would provide robust detailed information in biochemical analysis. We herein unveil a novel dual-mode optical (i.e. UV-vis absorption and fluorescence) method for multifunctional sensing of phosphate compounds (PCs) (e.g. nucleotides and pyrophosphate) based on pattern recognition, which innovatively employs only one kind of porphyrin/lanthanide-doped upconversion nanoparticles (Ln-UCNPs) hybrid integrated with a facile pH-regulated strategy as the sensor array. An easy-to-obtain porphyrin hydrate (tetraphenylporphyrin tetrasulfonic acid hydrate, TPPS) can assemble onto the ligand-free Ln-UCNPs to construct the organic/inorganic hybrid (TPPS/Ln-UCNPs), leading to a new absorption band to quench the upconversion fluorescence of Ln-UCNPs due to fluorescence resonance energy transfer (FRET). The dual-mode optical performances of TPPS/Ln-UCNPs are characteristically correlated with the pH in aqueous solution. Thus, as a proof-of-concept design, three types of TPPS/Ln-UCNPs (TPPS/Ln-UCNPs4, TPPS/Ln-UCNPs4.5 and TPPS/Ln-UCNPs5) were prepared by using buffers with different pH (at 4, 4.5 and 5) to form our proposed sensor array, which would result in individual dual-mode optical response patterns upon challenged with PCs for their pattern recognition through a competitive mechanism between TPPS and PCS. The results show that 3 TPPS/Ln-UCNPsn sensors can successfully permit the sensitive detection of 14 PCs and differentiate them between different concentrations, as well as a mixture of them. The pH-dependent TPPS/Ln-UCNPs promises the simple, yet powerful discrimination of PCs via pattern recognition, would prospectively stimulate and expand the use of organic/inorganic hybrid toward more biosensing applications.

Introduction Large-scale identification of phosphate compounds (PCs), such as nucleotides, pyrophosphate (PPi) and phosphate (Pi), has drawn more and more recent attention, as they constitute the essential units of all life forms.1 For example, adenosine triphosphate (ATP), as the crucial carrier of chemical energy in biological species, is momentous substrate in living organisms, playing a key role in major enzymatic activities.2 The dissipative and concentration rate of ATP are closely related with numerous diseases, such as hypoglycemia, hypoxia, Parkinson’s disease, ischemia and some malignant tumors.3 Moreover, ATP is an indicator for cell injury and cell viability.4 Guanosine-5′-triphosphate (GTP) participates in the synthesis of RNA, DNA and proteins, and cell signaling as well as in nutrient metabolism.5 PPi, the product of ATP hydrolysis under cellular conditions, is a biologically important target involved in DNA replication catalyzed by DNA polymerase.6 Based on the above facts, the facile detection and discrimination of those PCs is therefore highly important in clinic diagnosis and biochemical study. Various strategies have been reported for the assay to PCs, including capillary electrophoresis, aptamer-based optical sensing, enzymatic assays, mass spectrometry and electrochemical biosensors.7-12 Although each strategy has unique merits, each also more or less presents its deficiencies. These approaches may be subject to inherent limitations of high cost, high background, tedious synthesis steps and a narrow range of relatively high detection concentrations. The traditional sensing method, such as antibody-/aptamer-based ‘lock-key’ specific recognition, often work by using one sensor toward per analyte, thus demanding intensive labor to develop analyte-specific sensors. With this issue in mind, great efforts have been dedicated to introduce pattern identification for designing array or differential sensors termed ‘chemical noses/tongues’, which can discriminate multiple analytes by

exploring the integrated pattern responses from a set of sensing elements. This strategy has been reported for distinguishing metal ions with chemosensors,13-15 proteins with dye-labeled ensemble aptamers,16 and serum proteins with protein−nanoparticle conjugates.17 Array-based sensors can therefore discriminate multiple analytes with high accuracy and throughput,18-20 but few attempts have been reported for PCs. Accordingly, the development of simple, sensitive, rapid and novel strategies for large-scale identification and classification of PCs is highly desirable. Lanthanide-doped nanoparticles have been mined in numerous fields due to their alluring properties, especially time-resolved and/or upconversion fluorescence.21-30 Lanthanide-doped upconversion nanoparticles (Ln-UCNPs) can typically convert a NIR long wavelength radiation to a visible short wavelength fluorescence based on a two-photon or multiphoton mechanism, thus having appealing merits including narrow emission peak, low photobleaching, large Stokes shifts, etc. Ln-UCNPs-based fluorescent assays are widely developed based on fluorescence resonance energy transfer (FRET) mechanism for distinct analytes.31-33 However, there are rare reports about Ln-UCNPs-based FRET assays to discriminate PCs. Moreover, Ln-UCNPs-based dual-mode multifunctional assays to PCs remain unexplored. Inspired by the above facts, we herein for the first time demonstrate a novel, dual-mode optical sensor array for multifunctional identify and detect 14 PCs (nucleotides: ATP, ADP, AMP, CTP, CDP, CMP, GTP, GDP, GMP, UTP, UDP, UMP; PPi, and Pi), taking advantage of pH-regulated optical performances in organic/inorganic hybrid consisting of one porphyrin (TPPS) and Ln-UCNPs (Scheme 1). Firstly, the oleic acid (OA)-capped β-NaYF4: 20%Yb, 0.5%Tm Ln-UCNPs were prepared by using a solvothermal method,31 which is treated with HCl at pH 4 for the release of oleic acid from the surface, resulting in water-dispersible ligand-free

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Ln-UCNPs.34 Secondly, the as-prepared Ln-UCNPs were mixed with TPPS to form the hybrid of TPPS/Ln-UCNPs due to the interaction of TPPS and lanthanide ions at Ln-UCNPs’ surface, producing a dual-mode optical output, in which TPPS would show a new absorption band to quench the upconversion fluorescence of Ln-UCNPs via FRET. The resulting TPPS/Ln-UCNPs can be regulated by pH (e.g. pH 4, 4.5 and 5) to display distinct optical performances regarding UV-vis absorption and fluorescence, which can be used to develop a dual-mode sensor array (i.e. TPPS/Ln-UCNPs4, TPPS/Ln-UCNPs4.5, and TPPS/Ln-UCNPs5) for pattern recognition-based biosensing. As a proof-of-concept design, these 14 PCs were chosen as the model analytes. For PCs, their phosphate groups interact extensively with metal ions through Lewis acid/base interaction.35 Meanwhile, the nucleobases of nucleotides also have different metal-binding sites with various affinities.36 Thus, PCs can firmly combine with the lanthanide ions at Ln-UCNPs’ surface, resulting in the replacement of TPPS from TPPS/Ln-UCNPs via a competitive mechanism. With the respective addition of PCs into TPPS/Ln-UCNPsn sensors, they can present varying optical response patterns due to their diverse affinities towards PCs, which can be readily analyzed by principal component analysis (PCA) for pattern recognition of PCs indicated. Moreover, in a blind test, all PCs are successfully confirmed with an accuracy of 100%. Our TPPS/Ln-UCNPsn sensor array is further applied to detect PCs in non-invasive biofluids including urine and saliva, promises its real practical usage with great accuracy and robustness. Scheme 1. Schematic Illustration of the pH-Regulated Organic/Inorganic Hybrid Sensor Array for Dual-Mode Optical Pattern Discrimination of Phosphate Compounds (PCs).

Experimental Section Chemicals. Tetraphenylporphyrin tetrasulfonic acid hydrate (TPPS) was purchased from TCI Co. Ltd (Shanghai, China). Lanthanide metal salts (YCl3⋅6H2O, YbCl3⋅6H2O and TmCl3⋅6H2O, 99.99%) were ordered from Diyang Chemical Co. Ltd (Shanghai, China). Oleic acid (OA, 90%), 1-Octadecene (ODE, 90%) were purchased from Sigma-Aldrich (St. Louis, MO). Sodium hydroxide (NaOH), ammonium fluoride (NH4F), sodium pyrophosphate (Na4P2O7, denoted as PPi), sodium phosphate (Na3PO4, denoted as Pi) and metallic salts were purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). Adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), uridine triphosphate (UTP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), cytidine diphosphate (CDP), cytidine monophosphate (CMP), guanosine diphosphate (GDP), guanosine monophosphate (GMP), uridine diphosphate (UDP) and uridine

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monophosphate (UMP) were purchased from Shanghai Sangon Biotechnology Co. Ltd. Acetate buffers with various pH (20 mM, pH=4.0, 4.5, 5.0) were prepared using sodium acetate and acetic acid in distilled water. Apparatus. The upconversion fluorescence spectra were recorded using a fluorescence spectrophotometer (Hitachi F7000, Japan) coupled with a 980-nm laser accessory. UV-vis absorption spectra were measured on a microtiter plate reader (infinite M200 pro, TECAN, Switzerland) using a Corning 96-well plate. Transmission electron microscopy (TEM) images were taken on a JEOL-2100F electron microscope (JEOL, Tokyo, Japan). Fourier transform infrared spectroscopy (FTIR) spectra were obtained with a Nexus 670 optical bench (Nicolet, USA). Zeta potential characterizations were performed on a Malvern Instruments Zetasizer Nano-ZS instrument. Synthesis of oleic acid (OA)-capped lanthanide-doped upconversion nanoparticles (OA-Ln-UCNPs). OA-capped NaYF4: 20%Yb, 0.5%Tm Ln-UCNPs were prepared referring to the reported solvothermal method with minor modification.31 Detailed information about the preparation of OA-Ln-UCNPs is present in the Supplementary Information. Preparation of water-dispersible ligand-free lanthanide-doped upconversion nanoparticles (Ln-UCNPs). The water-dispersible ligand-free β-NaYF4: 20%Yb, 0.5%Tm Ln-UCNPs were prepared according to the reported literature.34 More introduction about the preparation of Ln-UCNPs is present in the Supplementary Information. Dual-mode optical pattern recognition of PCs via the pH-regulated TPPS/Ln-UCNPs sensor array. The TPPS/Ln-UCNPs sensor array was prepared by respectively mixing TPPS and Ln-UCNPs in 20 mM acetate buffer with different pH at 4.0, 4.5, and 5.0 (viz. TPPS/Ln-UCNPs4, TPPS/Ln-UCNPs4.5, TPPS/Ln-UCNPs5), and the mixture was incubated for 5 min at room temperature. The final concentrations of TPPS and Ln-UCNPs were 15 µM and 0.1 mg/mL, respectively. For dual-mode optical discrimination of 14 PCs (ATP, ADP, AMP, CTP, CDP, CMP, GTP, GDP, GMP, UTP, UDP, UMP, PPi, and Pi), an aliquot of the test PCs or distilled water (as the blank sample) was added to the TPPS/Ln-UCNPs sensor array. Then the mixture was incubated for 20 min before the optical measurements (UV-vis absorption and fluorescence spectra, respectively). Data processing. Principal component analysis (PCA) was conducted by using SPSS 22.0 software (IBM). Each sample was repeated in quintuplicate. The plots and heat maps were processed via GraphPad Prism 7.0 software (San Diego, CA, U.S.A.).

Results and Discussion Characterization of Ln-UCNPs. Oleic acid (OA)-capped lanthanide-doped upconversion nanoparticles (OA-Ln-UCNPs) were prepared using the reported solvothermal method with minor modification. After centrifugal/washing purification, The OA-Ln-UCNPs dispersed in chloroform were subjected to a simple acid treatment for the removal of OA ligand to acquire water-dispersible ligand-free Ln-UCNPs. From the FITR spectra, there is no characteristic bands of OA in Ln-UCNPs, proving the eliminating of OA ligand via the acid treatment (Figure S1). TEM images show the morphology of Ln-UCNPs almost maintain the same after the acid treatment with ligand removal (Figure S2). As depicted in Figure S3, the upconversion fluorescence feature of Ln-UCNPs in water are


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Analytical Chemistry like OA-Ln-UCNPs in chloroform upon excited with 980 nm laser, displaying the same representative emission peaks at around 455 nm and 480 nm respectively assigned to the dopant Tm3+ transitions of 1D2→3F4 and 1G4→3H6. The resultant Ln-UCNPs can be further used to combine with functional organic ligands to configure organic/inorganic hybrid. Investigation of interfacial interaction and design of TPPS/Ln-UCNPs sensor array for PCs. Porphyrins, the pigments of life, are a kind of heterocyclic macrocyclic compounds with their inherent plentiful coordination abilities and optical properties, which play a vital role in various fields such as materials, medicine and catalysis,37 and potentially suit as candidate ligands for the development of organic/inorganic hybrid sensors. In this work, TPPS, a commercially available water-soluble porphyrin derivative, was chosen as a model ligand to integrate with Ln-UCNPs for the construction of organic/inorganic hybrid as a dual-mode optical sensor array for discriminating PCs via our proposed pH-regulated strategy.

Figure 1. UV-vis absorption spectra and upconversion fluorescence emission of TPPS, Ln-UCNPs, TPPS/Ln-UCNPs, and TPPS/Ln-UCNPs+ATP in the acetate buffer with pH at 4.0.

TPPS contains four sulfo groups, which can coordinate with the lanthanide ions at the surface of Ln-UCNPs. The zeta potential of TPPS and Ln-UCNPs were measured in the acetate buffer (20 mM, pH = 4) and the values are -15.90 mV and 27.73 mV, respectively (Figure S4). Thus, TPPS can unite with Ln-UCNPs to create an organic/inorganic hybrid (TPPS/Ln-UCNP) by both lanthanide/sulfo coordination and electrostatic interaction in acidic buffer condition,31 leading to a FRET between TPPS (as acceptor) and Ln-UCNPs (as donor) as demonstrated in Figure 1. Namely, there can emerge a new absorption band around 460-510 nm (purple curve) in the resultant hybrid of TPPS/Ln-UCNPs, which mostly overlaps with Ln-UCNPs’ emission from 450 nm to 520 nm (red curve) to quench the upconversion fluorescence via FRET (light green curve). In this regard, some ligands with stronger coordination properties may interestingly replace TPPS via a competitive way, which would induce the blocking of FRET and a fluorescence recovery of Ln-UCNPs for biosensing. As a proof-of-concept testing, the resulting TPPS/Ln-UCNPs was challenged with adenosine triphosphate (ATP, one of PCs). From Figure 1, there are dual-mode optical responses of TPPS/Ln-UCNPs toward ATP with a decrease in absorption band around 460-510 nm (blue curve) and an increase in upconversion fluorescence from 450 nm to 520 nm (yellow curve). To better investigate the binding of Ln-UCNPs toward TPPS and PCs (ATP, ADP, AMP, CTP, CDP, CMP, GTP, GDP, GMP, UTP, UDP, UMP, PPi, and Pi), density functional theory (DFT) calculations were theoretically carried out using the Becke three-parameter hybrid functional (B3LYP) method38 to compare the interactions involved in Ln-UCNPs

and the oxygen of TPPS’s sulfo group, and Ln-UCNPs and the oxygen of PCs’ phosphate, respectively. The Mulliken charges and surface electrostatic potentials were obtained after several cycles of geometry optimization (Figure 2a). Ln3+ was reported to have tendency interacting with phosphate, and as shown in Table S1-S15, electric densities in ATP, ADP, AMP, CTP, CDP, CMP, GTP, GDP, GMP, UTP, UDP, UMP, PPi and Pi are higher than that of TPPS, indicating that these PCs can potentially release the TPPS and then bind to Ln-UCNPs. PCA as a powerful statistical technique,39 was further used to quantitatively assess the resultant electric density of O in PCs’ phosphate. The 2D PCA plot indicates that ATP, ADP, AMP, CTP, CDP, CMP, GTP, GDP, GMP, UTP, UDP, UMP, PPi and Pi have distinguishable electric density of O from each other (Figure 2b), proving the power for their discrimination by using a series of TPPS/Ln-UCNPs-based sensor elements.

Figure 2. (a) Molecular structures and its surface electrostatic potential graphs of 14 PCs (ATP, ADP, AMP, CTP, CDP, CMP, GTP, GDP, GMP, UTP, UDP, UMP, PPi, and Pi). (b) 2D canonical score plot for electric density of O in 14 PCs indicated.

According to the PCA sensing principle, the converting of distinguishable electric density of PCs to optical responses of TPPS/Ln-UCNPs, a TPPS/Ln-UCNPs-based sensor array is needed for discriminating between PCs by exploring their integrated pattern responses from the setting of sensing elements. The typical method for setting PCA sensing elements is using various functional materials14 or one material modified with different ligands.15 In this study, in view of the pH influence on the structural parameters of TPPS,37,40 we assumed that pH could be used to regulate the optical performances of TPPS/Ln-UCNPs to build a novel sensor array with effective optical response patterns upon exposed with analytes for their pattern recognition. As demonstrated in Figure 3, by using buffers with different pH (at 4, 4.5 and 5), sole Ln-UCNPs are resistant to the pH exposure (Figure 3c and 3d) and TPPS alone has a pH-dependent absorption increase around 500-530 nm resulting from the changes of its structural parameters (Figure 3a and 3b); while the hybrid of TPPS/Ln-UCNPs not only show a pH-dependent absorption decrease around 460-510 nm of TPPS, but also reverse previous TPPS’s quenching effect on Ln-UCNPs to give rise to a FRET-relevant pH-dependent upconversion fluorescence increase from 450 nm to 520 nm of Ln-UCNPs (Figure 3d and 3e). From the above results, the marriage of both various PCs’ electric density and pH-regulated dual-mode optical responses of TPPS/Ln-UCNPs (i.e., TPPS/Ln-UCNPs4, TPPS/Ln-UCNPs4.5 and TPPS/Ln-UCNPs5 as the PCA-based sensor array) would sprout a potential sensory method for distinguishing multiple PCs via pattern recognition.



Figure 3. UV-vis absorption spectra and the upconversion fluorescence emission of (a, b) TPPS, (c, d) Ln-UCNPs, and (e, f) TPPS/Ln-UCNPs in the buffers with pH at 4.0, 4.5 and 5.0, respectively.

Dual-mode optical pattern recognition of PCs via the pH-regulated TPPS/Ln-UCNPs sensor array. To illustrate our proposed method for pattern recognition of PCs, ATP, ADP, AMP, CTP, CDP, CMP, GTP, GDP, GMP, UTP, UDP, UMP, PPi, and Pi were applied to challenge with the pH-regulated TPPS/Ln-UCNPs sensor array. The sensing conditions, including the ratio of TPPS and Ln-UCNPs in the hybrid sensors, and reaction time were first optimized (see Supporting Information). As shown in Figure S5, the fluorescence of Ln-UCNPs can be quenched by the presence of increasing concentrations of TPPS via FRET in the hybrid of TPPS/Ln-UCNPs, and a saturated quenching efficiency can be reached when 0.1 mg/mL Ln-UCNPs encountered about 15 µM TPPS in buffers with pH at 4, 4.5 and 5, respectively. TPPS/Ln-UCNPs also showed a good stability of their dual optical performances in buffers with pH at 4, 4.5 and 5, respectively (Figure S6). The dual-mode optical responses of pH-regulated TPPS/Ln-UCNPs to ATP are rapid and can reach equilibrium within 20 min (Figure S7 and S8). To evaluate the precision of responses, 8 ATP replicates (20 µM) were challenged with TPPS/Ln-UCNPs at pH=4, 4.5 and 5, respectively, and the relative standard deviation (RSD) of UV-vis absorption responses was respectively 1.80%, 2.10%, and 1.50%, and the RSD of upconversion responses was respectively 1.78%, 1.93%, and 1.32%, indicating that TPPS/Ln-UCNPs sensor array has an excellent reproducibility. Unless noted otherwise, the following experiments were all performed under these conditions. The pH-regulated TPPS/Ln-UCNPs sensor array was then respectively incubated with 20 µM ATP, ADP, AMP, CTP, CDP, CMP, GTP, GDP, GMP, UTP, UDP, UMP, PPi, and Pi, and there were various dual-mode UV-vis absorption response [(A0-A)/A0] patterns and upconversion response [(F-F0)/F0] patterns toward different PCs due to the differentiable competitions between TPPS and PCs indicated (Figure 4a and 4b), where A0 and A represent the UV-vis absorption intensities in the absence and the presence of PCs, and F0 and F represent the upconversion fluorescence intensities in the absence and the presence of PCs, respectively. Five replicates of dual-mode optical response patterns of the pH-regulated TPPS/Ln-UCNPs sensor array toward PCs were recorded and then subjected to PCA to gain the responding canonical factors for pattern recognition of each PC, which represent linear combinations of optical response matrix (three

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TPPS/Ln-UCNPs sensors × number of PCs × five replicates). The two most significant canonical factors were employed to process 2D PCA plots (Figure 4c and 4g), in which each point means the response pattern for an individual PC sample against our proposed TPPS/Ln-UCNPs sensor array. As revealed in Figure 4c, the 70 canonical UV-vis absorption response patterns (14 PCs × 5 replicates) can be classified into varied groups. Moreover, the 70 canonical upconversion fluorescence response patterns (14 PCs × 5 replicates) can be also clustered into diverse groups in Figure 4g. Hence, these 14 PCs can be successfully differentiated with dual-mode optical outputs by using the pH-regulated TPPS/Ln-UCNPs pattern recognition system, confirming the feasibility of our pH-regulated strategy discussed above. Because the second discriminant factor (PC2) in the PCA plots was far less than 40%,14 it is acceptable to use the first discriminant factor (PC1) to profile the patterns of PCs (Figure 4d and 4h), the results can provide a direct traceable linear correlation between structurally similar PCs (e.g. ATP, ADP, AMP). Noticeably, to the best of our knowledge, this is the first report about using a pH-regulated organic/inorganic hybrid sensor array for the simple yet large-scale discrimination of 14 PCs. In this work, we also investigated whether the presence of high concentration of Cu2+ poses interference on the present system.31 As demonstrated in Figure S9, the high concentration of Cu2+ can be well separated from PCs indicated, which clearly certifies that the pH-regulated TPPS/Ln-UCNPs sensor array is powerful and robust for the discrimination of PCs.

Figure 4. (a) UV-vis absorbance and (e) upconversion fluorescence response patterns of pH-regulated TPPS/Ln-UCNPs sensor array toward 20 µM PCs (ATP, ADP, AMP, CTP, CDP, CMP, GTP, GDP, GMP, UTP, UDP, UMP, PPi and Pi). Heat maps derived from the (b) UV-vis absorbance and (f) upconversion fluorescence response patterns of pH-regulated TPPS/Ln-UCNPs sensor array toward 20 µM PCs indicated. Canonical score plots for (c) UV-vis absorbance and (g) upconversion fluorescence response patterns obtained with pH-regulated TPPS/Ln-UCNPs sensor array toward 20 µM PCs indicated. Plot of the first discriminant factor (PC1) for (d) UV-vis absorbance and (h) upconversion fluorescence response patterns toward 20 µM PCs indicated.

To evaluate the sensitivity of the pH-regulated TPPS/Ln-UCNPs sensor array, ATP and PPi were utilized to determine its analytical performances (Figure 5a-5d, S10, and S11). Since PC2 in the PCA plots was smaller than 40%, it is also acceptable to employ PC1 to correlate the concentrations of ATP (Figure 5b and 5d) and PPi (Figure S11d and S11h). More importantly, this dual-mode optical sensory platform not only demonstrates its excellent identification ability, but also provides a favorable sensitive discrimination of ATP (Figure


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Analytical Chemistry 5b and 5d) and PPi (Figure S11d and S11h). The detection limit of ATP in UV-vis absorption mode and fluorescence mode were 0.052 µM and 0.066 µM, respectively. The detection limit of PPi in UV-vis absorption mode and fluorescence mode were 0.20 µM and 0.36 µM, respectively. Further experiments demonstrated that the pattern recognition capability of the pH-regulated TPPS/Ln-UCNPs sensing system is equally effective in the discrimination of 20 µM CTP and ATP mixture with CTP/ATP molar ratios of 20/0, 15/5, 10/10, and 5/15, 0/20 (Figure 5e, 5f and S12), and the discrimination of 20 µM PPi and Pi mixture with PPi/Pi molar ratios of 20/0, 15/5, 10/10, and 5/15, 0/20 (Figure S13). Regarding UV-vis absorption and upconversion fluorescence response patterns, the mixtures of ATP and CTP with different molar ratios, and the mixtures of PPi and Pi with different molar ratios are clearly distinct in the PCA plots with outstanding performances. Additionally, to examine the reliability of the pH-regulated TPPS/Ln-UCNPs sensor array, unknown samples were tested and randomly taken from the training set, whose identification accuracy was found to be 100% (Table S16 and S17).

+ 15 µM CTP (Note: A and B are in UV-vis absorption mode to human urine); (C) 5 µM ATP + 15 µM CTP; (D) 15 µM ATP + 5 µM CTP (Note: C and D are in upconversion fluorescence mode to human urine); (E) 15 µM ATP + 5 µM CTP; (F) 5 µM ATP + 15 µM CTP (Note: E and F are in UV-vis absorption mode to human saliva); (G) 5 µM ATP + 15 µM CTP; (H) 15 µM ATP + 5 µM CTP (Note: G and H are in upconversion fluorescence mode to human saliva). The results of PCA plots indicate that the test samples can be observed in the right position agreeing with their ratios (Figure 6). For instance, sample A was shown between the two standards with ATP/CTP molar ratios at 20:0 and 10:10, respectively. This location demonstrates that it contains 10-20 µM ATP and 0-10 µM CTP, which is well consistent with the results provided by a single-mode reference assay based on the characteristic absorption of ATP/CTP around 260-280 nm. Thus, the present pH-regulated TPPS/Ln-UCNPs dual-mode optical sensor array has a great promise in practical application (e.g. non-invasive diagnosis of PCs-relevant diseases) with great accuracy and reliability.

Figure 5. Identification of ATP at various concentrations via the pH-regulated TPPS/Ln-UCNPs sensor array. Canonical score plot for (a) UV-vis absorption response patterns and (c) upconversion fluorescence response patterns obtained with the pH-regulated TPPS/Ln-UCNPs sensor array toward different concentrations of ATP. Plots of PC1 vs the concentrations of ATP in (b) UV-vis absorption mode and (d) upconversion fluorescence mode. Canonical score plot for the pH-regulated TPPS/Ln-UCNPs sensor array toward mixtures of CTP and ATP with different molar ratios in (e) UV-vis absorption mode and (f) upconversion fluorescence mode. The level of biofluid-derived PCs closely interrelates with human health,41 making them attractive model analytes for non-invasive health monitoring. To guarantee that the present system can be applied in accessible biofluids (such as human urine and saliva), we prepared a series of test samples. For UV-vis absorption mode, one set of standard samples was prepared by spiking ATP and CTP at different molar ratios (ATP/CTP= 20:0, 10:10, 0:20, all in µM) to human urine and saliva; for upconversion fluorescence mode, the other set of standard samples was formed by spiking ATP and CTP at different molar ratios (ATP/CTP= 0:20, 10:10, 20:0, all in µM) to human urine and saliva. Eight test samples were prepared for comparison: (A) 15 µM ATP + 5 µM CTP; (B) 5 µM ATP

Figure 6. Analysis of ATP/CTP mixture in human urine and saliva. Canonical score plots for (a, c) UV-vis absorption and (b, d) upconversion fluorescence response patterns obtained with the pH-regulated TPPS/Ln-UCNPs sensor array in test biofluid samples. The detailed information was listed in the table. *Note: a single-mode absorption assay based the characteristic absorption of ATP/CTP at 260-280 nm was used a reference.

In summary, we have developed a novel pH-regulated TPPS/Ln-UCNPs dual-mode optical sensor array for the distinguishiment of PCs utilizing a competitive mechianism between TPPS and PCs based on pattern recongition. There are several appealing features in this TPPS/Ln-UCNPs sensory platform in the view of easy-to-operation, low-cost,



quick-response time, and flexibility. This work opens a smart strategy to regulate the optical performances of organic/inorgainc hybrid for dual-mode pattern recognition-based biosensing and would promise new opportunities for the development of multifunctional sensing platforms for various applications.

Supporting Information Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Detailed information about the preparation of Ln-UCNPs, and their characterizations regarding FITR, TEM, upconversion fluorescence spectra, zeta potential; the optimization of reaction conditions; UV-vis absorbance and upconversion fluorescence response patterns of the pH-regulated TPPS/Ln-UCNPs sensor array toward nucleotides, different concentrations of ATP, a mixture of ATP and CTP; Tables of Mulliken charges in TPPS, and nucleotides; Identification of unknown nucleotides using the pH-regulated TPPS/Ln-UCNPs dual-mode optical sensor array

AUTHOR INFORMATION Corresponding Author *Min Zhang, Email: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21775044, 21675053, 21635003).

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For TOC only


Inorganic Hybrid: A

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