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Dual-functional carbon dots pattern on paper chips for Fe3+ and ferritin analysis in whole blood Shan-Wen Hu, Shu Qiao, Bi-Yi Xu, Xiang Peng, Jing-Juan Xu, and Hong-Yuan Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04891 • Publication Date (Web): 27 Dec 2016 Downloaded from http://pubs.acs.org on December 27, 2016

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Analytical Chemistry

Dual-functional carbon dots pattern on paper chips for Fe3+ and ferritin analysis in whole blood Shan-Wen Hu, Shu Qiao, Bi-Yi Xu*, Xiang Peng, Jing-Juan Xu*, Hong-Yuan Chen

State Key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, PR China

* Corresponding author. Tel/Fax: +86-25-89687294; E-mail address: [email protected] (J.J. Xu); [email protected] (B.Y. Xu)

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ABSTRACT:

Though microfluidic paper analytical devices (µPADs) have attracted paramounting attentions in recent years as promising devices for low cost point-of-care tests, their real applications for blood analysis are still challenged by integrating sample preparation with different detection modes on a same µPAD. Herein, we developed a novel µPAD, which well coupled automatic serum extraction with reliable dual mode iron health tests: fluorescent analysis for Fe3+ and colorimetric ELISA for ferritin. All these functions are made available by in-situ carbon dots (CDs) and AuNPs sequential patterning techniques. For CDs immobilization, hydrothermal reaction was taken on paper, to which a patterned through-hole polydimethylsiloxane (PDMS) mask was applied. None fluorescence CDs (nF-CDs) were generated on exposed regions, while the fluorescent CDs (F-CDs) were generated simultaneously on covered regions. Sensitive serum iron quantification was realized on the F-CDs modified regions, where Fe3+ ion can selectively quench the fluorescence of F-CDs. For AuNPs immobilization, electroless plating was taken on nF-CDs modified regions. The resulting AuNPs on nF-CDs layer on one hand triggered the coagulation of blood cells and thus led to the longest ever wicking distance for serum separation, on the other hand facilitated colorimetric enzyme linked immunosorbent assay (ELISA) for detection of serum ferritin. Combining the two readings, the µPAD can provide reliable measurement for serum iron and serum ferritin in whole blood. Furthermore, as CDs and AuNPs modified µPAD has the features of easy handling, low-cost, lightweight and disposability, it is accounting for a promising prototype for whole blood point-of-care analysis.

KEYWORDS: microfluidic paper chip, carbon dots, whole blood, Fe3+analysis, ferritin analysis

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INTRODUCTION

Microfluidic paper analytical devices (µPADs) have experienced booming development over the past decade due to their promising characters: inexpensive, easy to fabricate, user-friendly, portable and disposable.1, 2 All these specialties come from the nature of paper.3, 4 Firstly, because the device is mainly composed of paper, it is cheap in price and relatively easy for shaping, patterning and reagent immobilization. Secondly, the capillarity of the paper provides enough power to motivate the flow on the chip, requires for no complex external pumps. 5 Thus, µPADs are attracting paramounting attentions for applications in health monitoring, environment protection and food industry.6 However, there are still large room for future development of µPADs as for the range of adaptable analytical principles, the ability for multiplex and multi-principle analysis and the versatility to couple pre-treatment and analysis. To expand the analytical principles adaptable on µPADs, the major challenge comes from integrating functional reagents to the substrate. Besides tests that require for no washing steps, most analyzing principles with high selectivity and sensitivity would require recognition molecules to be linked with the substrate strong enough to withstand rigorous washing. Typically, there are three ways to form strong linkage between substrate and the reagent: adopt special substrate that can strongly adsorb target reagent4, perform chemical reactions to covalently link the reagent molecule to the substrate7 or interface the molecular and the surface of the substrate with nanomaterials8-15. In this paper, we focus on nanomaterial modification. Considering the outstanding physical and chemical characters of nanomaterials that have received wide recognitions in the fields of biosensing, nanomaterial modification might introduce large potential to increase the versatility of µPADs8. Au9-12, Carbon dots (CDs)

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and many other nanomaterials8 have all been successfully applied for constructing µPADs.

However, the coupling of nanomaterial to paper is generally limited to chemical deposition of Au and complex materials with it.

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Thus it is highly valuable to develop novel techniques to introduce

other functional nanomaterials to µPADs.


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Another challenge for µPADs is to carry out multiplex and even multi-principle analysis, because in real applications, the information required for diagnostics is usually not simplex. Several pioneering works have successfully integrated multiplex analysis on µPADs, which can give out results for a group of similar targets.4, 18 In some cases, different targets need to be analyzed with different principles, so it is also worthy to integrate different analyzing principles on a same µPAD. Thus, patterning methods that can define spatially different functional regions need to be developed. The third challenge we always meet is for functional integration of sample preparation with analysis, especially for in-situ analysis of complex samples like whole blood19-23. For whole blood, usually centrifugation is required before deposited to µPADs to get rid of blood cells. Though the creative handheld centrifugation partly relieves this problem, it is still a separated part. 24 To integrate automatic blood cell separation on µPADs, either size exclusion21, 25-27 or immuno-agglutination28 can be adopted. For size exclusion effect，usually special channels or layers are integrated on chip so as to focus blood cells in channel and eventually get rid of them.

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While for immuno-agglutination, additional reagent

immobilization is required to trigger the cell agglutination reaction. Thus, there is still room for simpler solutions. In addition, improving the separation efficiency by means of increasing the wicking distance of the separated serum also deserves consideration, which is especially needed for taking multiplex analysis30. Herein we proposed a novel µPAD that could trigger automatic blood coagulation and realize both fluorescent analysis for serum iron and colorimetric detection for serum ferritin. The µPAD was fabricated by sequentially patterning the filter paper with in-situ growth of CDs and AuNPs. For CD insitu growth, a through-hole PDMS mask was applied onto the filter paper, which rendered different reaction rates for the paper and consequently led to two distinguished regions on paper simultaneously: the fluorescent CDs (F-CDs) modified region and the non-fluorescent CDs (nF-CDs) modified region. The F-CDs modified region could be sensitively and selectively quenched by serum iron, which was applied for serum iron analysis. Further chemical deposition of Au was taken on the nF-CDs modified region. The resulting surface showed strong triggering ability for blood coagulation, thus enabled ACS Paragon Plus Environment

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automatic and highly effective serum extraction on this platform. Further bio-modification on AuNPs enabled sensing of serum ferritin on this region. As a whole, the readings of serum iron and serum ferritin provide us reliable information about the health of iron metabolism.

EXPERIMENTAL SECTION Materials Boric acid (BA), ethylenediamine (EDA), chlorauric acid (HAuCl4), KHCO3, NHydroxysuccinimide (NHS), 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and 3-mercaptopropionic acid were analytically pure from Sigma and used as received without further purification. Phosphate buffered saline (PBS) was purchased from Keygen Biotech Ltd. Antibodies for ferritin, ferritin, biotin-streptavidin-Horseradish Peroxidase (HRP) and 3,3',5,5'-Tetramethylbenzidine (TMB) colorimetric reagents were provided by Sangon Co, Ltd. Doubly deionized water (DI water, 18.2 MΩ at 25 ºC) prepared by a Milli-Q (MQ) water system was used throughout all experiments. Sylgard 184 elastomer base and curing agent for PDMS were both purchased from Dow Corning (Midland, MI). Whatman quantitative filter paper (slow) was purchased from GE Healthcare Life Sciences (Tokyo, Japan). Equipment and apparatus

Oxygen plasma bonding was conducted by Omega plasma apparatus

(Omega, Suzhou, China). The fluorescence spectra were recorded on Hitachi F-7000 Fluorescence spectrometer. Transmission electron microscope (TEM, JEM-2100) and Dynamic Light Scattering (DLS, BI-200SM) were applied for dispersed nanoparticle characterization. Scanning electron microscope (SEM, JEOL S-4800) was used for paper morphology characterization. Fourier-transform infrared spectra (FTIR) were tested on NICOLET iS10 infrared spectrometer. The X-ray photoelectron spectroscopy (XPS) spectrum was recorded by ESCALB MK-II, (VG Co., England) (under a base pressure of 1×10-9 Torr using monochromatic Mg-Kα X-rays at hν =1253.6 eV.) Fluorescence photograph (grey-scale) were captured by Bio-rad XRS+ Imaging System. Chip design and fabrication

Scheme 1 demonstrated the fabrication, functionalization and

application process of the paper-based serum iron health test chip. As shown in Scheme 1A, to generate ACS Paragon Plus Environment

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distinguished patterns of F-CDs and nF-CDs on filter paper, PDMS mask layers were applied to form a closed structure to encapsulate filter paper. PDMS mask was fabricated as follows. Briefly, 4.5 g of 10:1 (precursor: curing agent) PDMS mixture was poured onto the mold, degassed and heated at 80 ºC for 0.5 h before peeled off the mold. It was applied as the top layer. A bare chip was then cut into the designed structure as bottom layer. The top and bottom layers of PDMS were then irreversibly bonded in the boundary region after oxygen plasma treatment at 190 W for 30 s. So that the paper was encapsulated in the PDMS jacket and was spatially differentiated into two regions: the exposed region that could contact with reagents directly and the covered region that was protected by PDMS and could contact with reagents only by diffusion. Then the hybrid was subject to hydrothermal reaction, where the reaction kittle was filled with 20 mL doubly deionized water containing 2.0 g BA and 2.0 mL EDA, and were heated to 160 ºC for 12 h. After thoroughly washed with DI water, the encapsulated paper was subject to AuNPs electroless plating on the exposed regions. Plating solution contained 12 mM HAuCl4 ﹒4H2O, 0.5 M KHCO3 and 25 mM glucose (pH=9.3). 31 Chemical plating was allowed to proceed at 40 ºC for 4 h with continuously mild shaking. Then, the patterned paper was separated from PDMS jacket and cut into shot unit bands 3.5 mm for width and 9 mm for length with equal area for two functional regions: F-CDs modified region and AuNPs/nF-CDs modified region. Finally, antibody denoted as Ab1 for ferritin was immobilized onto the AuNPs by the following typical covalent conjugation procedures32. 100 µL 10% 3-mercaptopropionic acid was added to paper and kept for 12 h. Then 200 µL EDC/NHS (40 mg/mL: 20 mg/mL) was used to activate carboxyl groups for 1 h at room temperature. Afterwards, 50 µL 10.0 µg/mL primary antibody was bonded by EDC/NHS reaction to AuNPs and incubated for 12 h at room temperature before washed thoroughly by PBS. A blocking buffer (0.05 % (v/v) Tween-20 and 1% (v/v) BSA in PBS) was added to paper and kept for 10 min before washing. Detection of Fe3+

To acquire standard curve for Fe3+, Fe3+ standard solutions with different

concentrations were added to paper and the fluorescence spectra of each sensing area were taken.


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Selectivity was confirmed by addition of Ca2+, K+, Mg2+, Na+, Pb2+, Cu2+, Fe2+ and Ag+ at concentration of 1 mM, 100 µM HNO3 and Sample S1 for fluorescence quenching test. Sample S1 is the supernatant of the following mixture, where 1 mL human blood sample was mixed with 2 mL red blood cell lysate buffer and incubated at room temperature for 5 min before addition of 100 µM ascorbic acid. Detection of ferritin Scheme 1B illustrates the typical steps of colorimetric ELISA for ferritin analysis on AuNPs/ nF-CDs region. Firstly, sample was added to paper and was washed away 1 h later, followed by biotin labeled secondary antibodies (50 µL, incubated for 30 min at room temperature), biotinstreptavidin-HRP (50 µL, incubated for 30 min) and TMB-based colorimetric read out (50 µL, incubated for 10 min). Artificial blood samples were prepared by adding predefined amount of ferritin to 10 mM PBS with blood cells. Simultaneous detection of Fe3+and ferritin in blood sample Typical process for simultaneous Fe3+ and ferritin analysis is illustrated in Scheme 1C. Firstly, 15 µL real blood of human with heparin, 50 µM citric acid and 100 µM HNO3 was loaded onto the µPAD,

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where the serum was extracted

automatically. Then the cell-free serum would diffuse to the AuNPs/nF-CD modified region and the FCDs region sequentially. After the serum was perfused to the F-CDs region, the fluorescence under 365 nm UV exposure was recorded and the degree of fluorescence quenching was analyzed for Fe3+ concentration quantification. Then, colorimetric ELISA was carried out on the AuNPs/nF-CDs region to measure the concentration of ferritin. Color change from pale red to dark blue was recorded and analyzed. RESULTS AND DISCUSSIONS Dual in-situ growth of both F-CDs and nF-CDs on paper CDs are drawing paramount attentions in recent years for their rich physical and chemical characteristics, low cytotoxicity, good water solubility and easy preparation.8 For µPAD development, many efforts have been paid to apply CDs on paper.5,14,36,37 However, there’s still no report for in-situ growth of CDs on paper which is advantageous


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because it not only can reduce the nanomaterials preparation steps and surface modification steps to a single step, but also can guarantee a strong linkage between the nanomaterial and the substrate. For in-situ growth of CDs on paper, hydrothermal reaction was taken, where filter paper and EDA served as carbon sources and BA as source for B-dopant. BA was selected to avoid the quenching phenomenon caused by CDs aggregation because the electron-deficient B atom could inhibit the intermolecular charge transfer between electron donors and acceptors.38 Constant sampling was carried out to examine the variation of the fluorescence along the growing process. Interestingly, as shown in Figure S1, the paper changed from dark to fluorescent over the first 30 min of in-situ growth, but fluorescence decreased over prolonged reaction time. This phenomenon implied that we had successfully realize in-situ growth of fluorescence materials on paper, and the fluorescence could be effectively tuned by controlling the growth time. To check if the resulting material on paper was exactly CDs, we characterized the floating material in the solution. Both TEM and fluorescence characterization confirmed the generation of CDs. In addition, FTIR tests shown in Figure S2 indicate the materials on paper had similar FTIR spectra with CDs in solution after freeze-dry, which confirmed that the material grown on paper was CDs. Moreover, absorption peaks standing for BO-H, B-O and amino group were also observable, which was evident for successful doping of boron to CDs on paper. Inspired by this phenomenon, we utilized a PDMS mask with through-holes to divide the paper into two different regions: the exposed region and the covered region. The paper was well sandwiched with the two PDMS layers during hydro-thermal reactions, which can be easily removed afterwards because the paper is not bonded with PDMS layers. The exposed region contacts with reagents directly and bears the same CDs growth speed as bare paper, while the covered region can only contact with reagents through diffusion and thus bears a much slower CDs growth speed. Figure S3 is the time-serial fluorescence profile for the two regions. These results proved that the evolution tendency for fluorescence on both exposed and covered regions conformed to that for bare paper, in which the fluorescence increased first and then diminished along the CDs growth process. However, the time required for the fluorescence to reach maximum is different for the two regions. For the exposed region ACS Paragon Plus Environment

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reaches maximum at 30 min, while that for the covered region occurs much later, which is 8 h. Based on this phenomenon, four stages were discernable for the fluorescence profile on patterned paper. In the first stage, no fluorescence could be observed both for the exposed and covered regions. Then, fluorescence appeared on the exposed region while the covered region remained dark. After about 1.0 h, fluorescence disappeared on the exposed region while it gradually increased on the covered region. At the last stage, after 72 h, the fluorescence on both regions was quenched. Both in the 2nd and 3rd stage the fluorescence contrast between the two regions could be evident. But considering the density of CDs available for later functionalization, 12 h in the 3rd stage was selected as the optimum condition for hydrothermal in-situ CDs patterning on paper. Figure 1A shows the typical image of the patterned paper fabricated under this optimized condition. Remarkable difference can be witnessed between F-CDs region and nF-CDs region, where blue fluorescence in the F-CDs regions reaches maximum, while that in the nF-CDs is generally non-observable, demonstrating an ideal patterning effect. To further delineate the difference between F-CDs region and nF-CDs region, both SEM geometrical characterization and XPS/EDX element content characterization were taken. In Figure 1B1-D1, the SEM images show the bare paper is smooth, and higher roughness is observable for the paper on CDs modified regions. The roughness for the nF-CDs modified region is even higher than that in the F-CDs modified region, implying a higher aggregation density for CDs in the nF-CDs modified region. Corresponding XPS is shown in Figure 1B2-D2. For all three samples, elements including carbon and oxygen are found on paper. But element boron only present on CDs modified papers. Moreover, the percentage of boron present in F-CDs (Figure 1C2) and nF-CDs (Figure 1D2) regions show clear differences, with the former reads 7.99% while the later 10.09%. EDX assays confirm the results of XPS. The detected boron content at F-CDs region is 8.87% while at nF-CDs region it is 10.47%. Since the content of boron is a direct reflection for CDs density on paper, the results imply the nF-CDs region has higher density of CDs than F-CDs region, which is in consistency with traditional theories for aggregation based fluorescence quenching of CDs. 38


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After successfully dual patterning of the substrate with CDs, we take a step further to examine the strength of the linkage between the materials and the substrate. Comparison was made between in-situ grown F-CDs on paper and the F-CDs dropwise-modified paper. They were immersed into water and examined repeatedly. As revealed in Figure S5, the fluorescence intensity of dropwise-modified paper declined and was totally vanished after 8 hours. Meanwhile fluorescence intensity of in-situ grown FCDs remained almost the same throughout the test. Even boiled in hot water for over 1 h, still no fluorescence quenching could be observed for in-situ grown CDs. In addition, their fluorescence stability over long time storage and different salt concentrations was also examined. As demonstrated in Figure S5c, less than 5% of intensity loss was found after 14 days, and even in 1 M NaCl, only 5.5% of intensity change could be observed than that in pure water. The results indicate the reported in-situ grown CDs has formed strong linkage with the paper substrate and is resistant to extreme conditions and persistent to long-term storage. AuNPs electroless plating on nF-CDs modified region

AuNPs are among the most widely applied

materials for biosensors because they are biocompatible, easy for linkage with functional molecules, and can be sensitive indicators when coupled with most of the powerful analytical principles all the way from colorimetry, fluorescence, Raman, electrochemistry and even mass spectrometry. Thus AuNPs have long been the dominant material for µPAD functional modification. So it is desirable to introduce AuNPs to empower the µPAD. In our experiment, electroless plating was applied for AuNPs deposition. Compared with plating time of over 12 h on bare paper, the plating time on nF-CDs modified paper is much faster: over 3 h of electroless plating, the nF-CDs modified paper become wine red, which implies AuNPs have been successfully grown on the surface. We also studied the process of AuNPs grown on paper both on the exposed and covered region. It is observed in the experiment that sufficient reagent is crucial to the AuNPs grown and lack of reagent will lead to slower speed and even cease of reaction. Hence we adopt this complex for in situ synthetize of AuNPs and stopped the reaction when abundant AuNPs can be generated on the AuNPs/nF-CDs region while barely no AuNPs can be observable on FCDs region. Figure S6 shown the resulting image of the final chip, it is observable that with the help of ACS Paragon Plus Environment

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through-hole PDMS mask, the growth of AuNPs can be well restricted on the exposed region. Figure 2A provides microscale details for the transient region. Three areas (A2-A4) as indicated by the red circles are selected for further inspection. Figure A2-A4 show the transient area from abundant AuNPs on nF-CDs regions to F-CDs modified region has a narrow width of less than 500 µm, confirming that this method can pattern AuNPs with acceptable spatial resolution. After geometrical characterization, we move on to test its ability for triggering blood coagulation because it is long known that rough surfaces can trigger and accelerate blood coagulation. However, the character have not been reported on µPADs, which might because the phenomenon is not strong enough on bare paper to facilitate automatic serum extraction and RBCs can easily penetrate through paper by deformation.39 Only those paper with pores smaller than 2.5 µm can separate plasma from RBCs in whole blood sample, yet the efficiency may not be sufficient. Till now, besides size exclusion with additional special films and immuno-agglutination, there’s no widely accepted mechanism that can be easily integrated for serum separation on µPADs.28, 40 Interestingly, on the proposed AuNPs modified nF-CDs paper, we found blood cell coagulation could be triggered automatically and with ideal efficiency. Figure 2B illustrates the coagulation phenomenon on different substrates. Figure 2B1 shows blood cell coagulation occurs on AuNPs/nF-CDs modified paper. Figure 2B2 as the magnified images further implies, the coagulation is fast enough for generating sharp boundaries for coagulated cells: at 0.5 mm from the dropping area only a few of residual was found, while at 1.0 mm away, no residual was found. The separation efficiency is also high, a droplet of 15 µL is enough to generate serum that can soak the proposed testing pad with area of 31.5 mm2 and wicking distance of 9.0 mm. Hence this AuNPs modified nF-CDs paper possesses the ability of serum separation from whole blood sample, which is in urgent need for resource limited applications. Figure 2B3 and 2B4 correspond to situation on nF-CDs modified region and the bear paper, where no obvious coagulation can be overserved, indicating that the blood cell coagulation is triggered by the AuNPs on the surface. To explore possible reasons, AuNPs plated paper was also prepared, which was able to trigger the coagulation, and it was


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observed that higher density of AuNPs would contribute to more prominent coagulation. Thus we can conclude, it is the presence of AuNPs that has facilitated the fast coagulation. Simultaneous analysis of serum iron and ferritin in blood sample

Iron metabolic abnormity is

among the key indicators to diseases like anemia, hepatitis, diabetes and cardiovascular diseases.33, 34, 41 Thus, handy and reliable iron health test is worthy for diagnostics, prognostics and treatment evaluation for these diseases. To examine iron health, both serum iron and serum ferritin need to be analyzed. However, since serum iron and ferritin could only be analyzed with different analyzing principles, their tests had hardly been integrated on a same platform before. The above proposed dual modification of paper chip relived the situation by providing us distinguish surfaces for coupling with two different analyzing principles. In the case for iron health test, the F-CDs region is adopted for fluorescence analysis of serum iron, and the AuNPs/nF-CDs region is functionalized for colorimetric immuneanalysis of serum ferritin. Analysis of serum iron is based on the quenching effect of Fe3+ for the fluorescence of the in-situ grown F-CDs. Figure 3A is a serial of fluorescence spectrums in F-CDs modified regions exposed to PBS solution spiked with different concentrations of Fe3+. The peak fluorescence intensity decreases with increment of Fe3+ concentrations. Figure 3B shows the corresponding standard curve for Fe3+ concentration over fluorescence intensity, which holds good linearity between 1 and 103 µM, well covered the physiological values available in human blood sample (between 9 and 32 µM). Besides linear range, we also examined the selectivity of the quenching effect. As shown in Figure 3C, addition of common ions including Ca2+, K+, Mg2+, Na+, Pb2+, Cu2+, Fe2+ and Ag+ all pose negligible influence to the fluorescence. In addition, the influence of all the other native factors besides Fe3+ in the blood was tested by reducing all the Fe3+ in the lysate blood samples with ascorbic acid. As a whole they contribute to nearly 2% of quenching effect for the total fluorescence intensity. Besides native factors, the influence of additive reagents also needs to be examined. To quantify the total amount of Fe2+ and Fe3+ from real sample, typically, heparin is added as anticoagulant; the citric acid is added to the blood sample so that all the bonded iron ion can be released; and HNO3 is added to oxide all the Fe2+ to the ACS Paragon Plus Environment

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Fe3+ so that all the iron ion can be counted. Since both heparin and citric acid were reported to have no influence to the quenching effect, we only examined the quenching effect of HNO3 to the fluorescence, where only near 1% fluorescence intensity decrement was observed. To analyze the ferritin in serum, immuno-colorimetry assay was executed. To eliminate the influence of native ferritin in real blood, artificial blood composed of blood cells in PBS was applied. Compared the colorimetric results of the control (Figure 4A) with that of the artificial blood with no spiked ferritin (Figure 4B), the color change is negligible, which implies the blood cells pose little interference to the colorimetric result. Figure 4C and 4E correspond to results for artificial blood spiked with 15 µg/L and 150 µg/L ferritin, which covered the range of serum ferritin for healthy ones. The results show with the increment of ferritin concentration, the color change from wine red to dark blue. Figure 4D and 4E are the results for real blood sample from healthy one and that spiked with 150 µg/L ferritin as pseudo patient sample. The results show the color for the healthy one is within the range of 15 µg/L and 150 µg/L, while the pseudo patient sample is beyond the range. This confirms that the proposed method is capable of semi-quantification of blood serum and distinguishing the patient sample from the healthy ones. In addition, we also examined the interference of HNO3, considering HNO3 might denature the ferritin in blood. 80 µg/L ferritin with 100 µM HNO3 was added to the artificial blood in a typical ELISA test, showing no obvious interference to the result of samples without HNO3. For simultaneous analysis of both serum iron and serum ferritin in real blood sample, we tested one real blood sample from healthy candidate and compared the results from standard methods and that from the paper chip. Serum iron in the blood sample was determined to be 27.3 ± 1.5 µM by paper chip. The accuracy of this method was confirmed by the standard addition method in Table 1. The ferritin value was determined to be 80.1 µg/L by typical ELISA test (the standard curve was shown in Figure S8), which agreed with the results of the paper-based platform.

CONCLUSIONS


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In conclusion, a novel method for dual-functional in-situ growth and patterning of F-CDs and nF-CDs on paper was successfully developed. When combined with Au electroless plating, it has facilitated a powerful µPAD for whole blood iron health tests. A paper-based sensory region for Fe3+ was fabricated since the fluorescence of CDs on paper could be efficiently and selectively quenched by Fe3+. Meanwhile, a colorimetric detection region for ferritin was functionalized with AuNPs based ELISA. Combining the two regions, blood serum extraction, serum iron and serum ferritin analysis from whole blood are realized on this µPADs, providing reliable implications for the health of iron metabolism. Considering the versatility of CDs and AuNPs as substrates for biosensing, the µPAD proposed here might lay a solid foundation for a whole family of µPADs for multiplex and multi-principle whole blood analysis in the near future. ACKNOWLEDGMENT This work was supported by the Ministry of Science and Technology Program of China (2016YFA0201200), the National Natural Science Foundation of China (21535003, 21505069), and Natural Science Foundation of Jiangsu Province (BK20140597).

Supporting Information Available: Characterization of CDs, fluorescence photograph of patterned papers and fitting curve of standard ELISA for ferritin as noted in the text. This information is available free of charge via the Internet at http://pubs.acs.org. REFERENCES (1) Martinez, A. W.; Phillips, S. T.; Butte, M. J.; Whitesides, G. M. Angew. Chem., Int. Ed. 2007, 46, 1318. (2) Cunningham, J. C.; DeGregory, P. R.; Crooks, R. M. ;Bohn, P. W., Pemberton, J. E., Annu. Rev. Anal. Chem. 2016, 9, 183. (3) Martinez, A. W.; Phillips, S. T.; Whitesides, G. M.; Carrilho, E. Anal. Chem. 2010, 82, 3.


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Scheme Caption Scheme 1. Demonstration for µPAD fabrication and functioning. A. µPAD fabrication process. B. operation process of colorimetric ELISA for ferritin analysis; C. chip functioning process.

Figure Captions Figure 1. A. Paper sample under 365 nm UV light, left sample is bare paper, the right sample is the dual CDs patterned paper. SEM images of: B1. bare Paper, C1. F-CDs region, D1. nF-CDs regions. XPS results of: B2. bare paper, C2. F-CDs, D2. nF-CDs. Figure 2. SEM characterization of. A. images of AuNPs/nF-CDs modified paper with AuNPs at boundary area. A1. General view；A2-A4. Magnified view of the corresponding regions; B. Papers after loaded with whole blood sample; B1. AuNPs/nF-CDs modified paper after blood sample (3 µL) loaded; B2. Magnified image for B1; B3.SEM image of F-CDs modified paper after blood sample (3 µL) loaded. B4. SEM image of bare paper after blood sample (3 µL) loaded. Figure 3. Fluorescence spectrum of CDs A. on paper with different concentrations of Fe3+. B. Standard curve for relations between CDs fluorescence intensity and the concentrations of Fe3+. C. Selectivity for Fe3+ quenching over the fluorescence of CDs. S1 stands for blood samples with lysed red blood cells but no Fe3+ ions.


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Figure 4. Colorimetric results of A. control; B. Artificial blood with no ferritin; C. artificial blood with 15 µg/L ferritin; D. real blood sample; E. artificial blood with 150 µg/L ferritin; F. real blood added with 150 µg/L serum ferritin

Tables Table 1. Results for Fe3+ in blood samples.


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Scheme 1

Figure 1


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Figure 2

Figure 3


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Figure 4

Table 1 Total added(µM)

Determined(µM)

Recovery (%)

0

27.3±1.5

50

77.0±6.1

99.4

100

123.4±9.4

92.9

150

173.8±19.2

100.7

For Table of Contents Only


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and Ferritin Analysis in Whole Blood - ACS Publications - American

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