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A multiplexed SERS-active microneedle for simultaneous redox potential and pH measurements in rat joints Chenyan Pan, Xiaochen Li, Jie Sun, Zhe Li, Li Zhang, Weiping Qian, Peimin Wang, and Jian Dong ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00117 • Publication Date (Web): 01 Apr 2019 Downloaded from http://pubs.acs.org on April 6, 2019

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ACS Applied Bio Materials

A multiplexed SERS-active microneedle for simultaneous redox potential and pH measurements in rat joints Chenyan Pan,a, ‡ Xiaochen Li,b, ‡ Jie Sun,a Zhe Li,a Li Zhang,b Weiping Qian,*,a Peimin Wang,*,b and Jian Dong *,a,c a

State Key Laboratory of Bioelectronics, School of Biological Science and Medical

Engineering, Southeast University, Nanjing, 210096, China. b Department of Orthopedics and Traumatology, Affiliated Hospital of Nanjing University of Traditional Chinese Medicine, Nanjing, 210029, China. c Laboratory of Environment and Biosafety, Research Institute of Southeast University in Suzhou, Suzhou,215123, China. KEYWORDS: SERS-active microneedle, multiplexed SERS detection, redox potential, pH, rat joint

ABSTRACT: To detect redox potential and pH simultaneously in rat joints, a surface-enhanced Raman scattering (SERS)-active microneedle was structured with two separate grooves containing redox-sensitive and pH-sensitive SERS probes, respectively. The multiplexed SERS-active microneedles brought the two SERS probes into muscles with minimal invasion to sense their redox status and pH in 5 min, and they also detected the dynamical evolution of redox status and pH in muscles. The multiplexed SERS-active microneedles were also inserted into rat joints to

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sense their redox status and pH, which lack flowable fluids. The strategy of one SERS probe in one groove would make SERS-active microneedles become a multiplexed analytical tool of minimally invasive sampling in vivo and direct Raman detection ex vivo, and the multiplexed SERS-active microneedles would become a versatile analytical tool to promote researches of biomedicines.

INTRODUCTION In organisms, Redox potential (or redox status), dynamically changing mainly dependent on the amounts of reactive oxygen species (ROS), reactive nitrogen species (RNS), antioxidant molecules, and corresponding enzymes, is related to many physiopathological processes such as cell differentiation, division, apoptosis, necrosis, etc.1-4 ROS and RNS are physiological signaling molecules in these processes,5,6 and it does not have specificity with a specific disease but the change in redox status indicates physiological or pathological status, such as arthritis and diabetes, in which the redox status is related with the inflammatory processes of arthritis and the initial and consequential damage of diabetes, respectively.7,8 Up to now, most analytical methods have been developed for detecting biological samplings ex vivo. These methods ex vivo have some disadvantages. For example, it is difficult for collecting liquid samples from tissues lacked flowable fluids, such as bone joint or cartilage. Besides, during sampling and detection processes, neither redox status nor a given oxidative or reductive molecule would keep their original states in tissues due to exposed to air. Therefore, it is necessary for developing an effective alternative to detect those.


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The Raman-based techniques can be performed in situ and real time, which are desirable tools for monitoring biological samples.9 Pioneering work had been carried out by attaching redoxsensitive molecules onto gold nanoparticles to structure surface-enhanced Raman scattering (SERS) probes and then, the SERS probes were introduced into cells to detect intracellular redox potential.10-12 However, it is still a great challenge for detecting redox potential in tissues,13,14 such as skin and muscle, let alone bone joint and cartilage. Obviously, to detect SERS signals in vivo, SERS-active materials should be introduced into tissues, usually by surgery or injection, however it is difficult either to collect distinguishable SERS signals or to take the SERS-active materials out.15-17 Acupuncture needles, used in Chinese traditional medicine, are minimally invasive into the body. In our previously reports, SERS-active microneedles were structured by adsorbed SERSactive materials on an acupuncture needle to achieve minimally invasive sampling in vivo and direct Raman detection ex vivo.18-20 Various ingenious design and fabrication made the SERSactive microneedles become ideal miniaturized SERS substrates to avoid adverse effects of common SERS substrates used in vivo and to solve application of SERS from the culture cells to living animals. However, a SERS-active microneedle only detected a single object, and could not detect two or more ones, which is a general question in detection using SERS substrates. It is necessary for developing a multiplexed detection in many researches. For example, redox potential is pH dependent, so gaining an accurate redox potential it is also necessary to simultaneously measure pH. Arthritis is a common inflammation disease and causes disability, which leads severe limitations in function. Inflammatory progression is closely related with the evolution of redox status,7 and direct detection of the redox potential (redox status) would be a simple method for


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predicting progression and prognosis of arthritis. Here, a multiplexed SERS-active microneedle would be structured to simultaneously detect redox potential and pH in rat joints, and SD rats and arthritis model ones were used to assess the SERS-active microneedle. As shown in illustration 1, two separate grooves were made on an acupuncture needle, and one groove contained redox-sensitive SERS probes and the other ones contained pH-sensitive SERS probes. When the SERS-active microneedles were inserted into rat joints, the two SERS probes can be brought into the joint to sense its redox potential and pH; when they were pulled out, the SERSactive microneedles were used to collected SERS spectra to indicate its redox potential and pH.

Illustration1. A multiplexed SERS-active microneedle was inserted into a rat joint for sensing its redox potential and pH in vivo and then, the SERS-active microneedle was pulled out for collecting SERS spectra ex vivo. MATERIALS AND METHODS Materials. Stainless-steel acupuncture needles of 260 μm in diameter were purchased from Suzhou Tianxie Acupuncture Instruments Co., Ltd. (China). Chloroauric acid tetrahydrate (HAuCl4), anthraquinone-2-carboxylic acid (AQ), 4-mercaptobenzoic acid (4-MBA), lactic acid,


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and N- acetyl-L-cysteine were purchased from Guoyao group (China). All other reagents used were analytical grade. Pure water used in the experiments was prepared by Milli-Q water from Milli-Q system (resistivity >18 M). Fabrication of a multiplexed SERS-active microneedle for detecting redox potential and pH simultaneously. The two separated grooves were electrochemically corroded similar as previous report,20 and a modified step is that PS layer was cut twice with a blade at 0.65 and 0.5 cm from the tip respectively, as electrochemical corrosion sites to form two grooves. The gold nanoshells (GNSs) with a diameter of 175 nm were used as SERS substrates to structure redoxsensitive SERS probes (AQ on GNSs) and pH-sensitive SERS probes (4-MBA on GNSs), as previous reports.21,22 In brief, In brief, Redox-sensitive SERS probes, in brief, were prepared by attaching anthraquinone-2-carboxylic acid to amino of cystamine on GNSs, in which anthraquinone-2-carboxylic acid was active with dicyclohexyl-carbodiimide and Nhydroxysuccinimide; pH-sensitive SERS probes were prepared by attaching 4-MBA on GNSs. The redox-sensitive SERS probes were added manually in the distal groove to the tip and pHsensitive SERS probes were in the proximal ones. After the added suspensions were dried the SERS probes out of the grooves were erased in the direction from the distal to the tip to avoid the pH-sensitive SERS probes falling into the groove of redox-sensitive SERS probes (the signal intensity of the pH-sensitive SERS probes is stronger than that of the redox-sensitive SERS probes, and if the pH-sensitive SERS probes falling into the groove of redox-sensitive SERS probes the SERS spectra of the former would cover that of the latter, but the inverse is not). Evaluation of the multiplexed SERS-active microneedle ex vivo and in vivo. First, the SERS-active microneedles were immersed into disodium hydrogen phosphate-citric acid buffers for 10 min to evaluate the response of the pH-sensitive SERS probes in the groove to pH and the


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dependence of the SERS spectra of redox-sensitive SERS probes in the groove to pH. Second, SERS-active microneedles were inserted into gastrocnemius muscle of rats to evaluate the response of their two SERS probes in grooves to the normal physiological environments. Finally, 0.4 mL of lactic acid (20 g/L in 0.9% saline) and 0.4 ml of N-acetyl-L-cysteine (10 g/L in 0.9% saline) were injected respectively into the gastrocnemius muscle of rats to simulate abnormally physiological environments (pH and redox potential) to evaluate the response of their two SERS probes in grooves. Simultaneous detection of redox potential and pH measurements in rat joints. SD rats (male, 200 ± 20 g) used were of the specific pathogen free (SPF) class, obtained from Nanjing Qinglongshan animal breeding grounds (China). All procedures (injecting sodium iodoacetate, inserting the SERS-active microneedle, and killing) were performed under isoﬂurane anesthesia with the permission of the Science and Technology Department of Jiangsu Province (SYXK 2016-0014). The inflamed joints were induced in SD rats by injecting 50 μL of sodium iodoacetate (20 mg/ml in 0.9% sterile saline) into the knees under the patellar ligament.23 As shown in illustration 1, at fifteenth day after inflamed, a SERS-active microneedle was inserted into normal or inflamed joints for 10 min to sense redox potential and pH simultaneously and then, the SERS-active microneedle was pulled out for collecting, in turn, SERS spectra of redoxsensitive and pH-sensitive SERS probes. After the SERS-active microneedles were pulled out, the rats were killed for sampling synovium and cartilage. The synovium and cartilage were first fixed in 10 % formalin solution (pH 7.2) for 2 days and then, the cartilages were decalcified for about 30 days. Specimens were dehydrated in a graded series of ethanol baths and in xylene,


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embedded in paraffin, and sectioned in 5 μm thick slices. Hematoxylin and Eosin (HE) staining was used to show the inflammation and damage of the synovium and cartilage. Characterization and Measurements. Zeiss ULTRA-plus scanning electron microscopy (SEM) was used to characterize the morphology of SERS-active microneedles. Renishaw Invia microRaman spectrometer was used to collect SERS spectra at room temperature (~20 °C). A 785 nm laser was used by using a 50× long working distance objective (NA=0.5). The extinction power and the acquisition time were 600 μW and 10 s in all measurements, respectively. The intensity ratios of the two SERS probes were calculated as previous reports.19,22 Briefly, SERSI1143 cm-1/SERSI1180 cm-1 was the ratio of the intensity values at 1143 cm-1 and 1180 cm-1 minus the intensity value at 1120 cm-1 (as background value), respectively; SERSI1606 cm-1 and SERSI1666 cm-1 was the ratio of the intensity values at 1606 cm-1 and 1666 cm-1 minus the intensity value at 1700 cm-1 (as background value), respectively. RESULTS AND DISCUSSION Fabrication of a multiplexed SERS-active microneedle for detecting redox potential and pH simultaneously. To protect SERS-active materials integrated on acupuncture needles, the strategies from direct PS-coated and microporous PS-coated to groove-loaded SERS-active materials were designed for structuring SERS-active microneedles.18-20 Here, to separately detect two SERS signals from a SERS-active microneedle, two separate grooves were corroded electrochemically on an acupuncture needle. Fig. 1A showed a SEM image of a typical acupuncture needle with two grooves, and the distance between the two grooves is about 0.15 cm. Considering the tradeoff between the long distance between the two grooves which can avoid adding manually two SERS probes in one groove and the short distance between them


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which can endow the SERS-active microneedle sense multi-parameters in a given tissue with a small size, the distance between two grooves on SERS-active microneedles was 0.15 cm. Fig. 1B is a magnified SEM image of a groove, which can protect GNSs loaded.

Figure 1. (A) A SEM image of an acupuncture needle with two electrochemically corroded grooves, (B) A magnified SEM image of a typical groove, (C) A SEM image of SERS probes in a groove, and (D) A magnified SEM image of SERS probes in a groove (the inset is a SEM image of GNSs for fabricating SERS probes). In previous reports, GNSs were adsorbed on the thiolated acupuncture needles to fabricate SERS-active microneedles and then, the SERS-active microneedles were immersed in solution to adsorbed probe molecules on GNSs.18,19 Here, two kind of SERS probes, redox-sensitive and pH-sensitive SERS probes, were first fabricated by adsorbing probe molecules on GNSs respectively and then, the two SERS probes were added manually onto separated grooves to form a multiplexed SERS-active microneedle for detecting redox potential and pH simultaneously. Fig. 1C is a SEM image of SERS probes loaded in a groove after erased. Fig. 1D


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is a magnified SEM image of the SERS probes loaded in a groove, and the SERS probes packed randomly on the groove due to natural drying of the added suspension. SERS spectra and their pH response of multiplexed SERS-active microneedles. Fig. 2A showed the SERS spectra of the two SERS probes on a SERS-active microneedle after the added suspensions of the two SERS probes were dried on grooves. The two SERS probes were based on the same GNSs, but the SERS intensity of characterized Raman peaks of 4-MBA was higher about 7 times than that of AQ. If the two SERS probes were in one groove, the signal of 4-MBA would cover that of AQ in collected SERS spectra. The strategy of one SERS probe in one groove effectively solved the question of overlap or coverup among SERS spectra. Fig. 2B is the SERS intensity values of characterized Raman peaks of the two SERS probes of SERS-active microneedles. Because the SERS intensity of molecules on SERS substrates was influenced by the repeatability of SERS substrates, the number amount of the molecules, and the environment of the molecules, the collected SERS intensity on most SERS substrates have a high variability. Ratiometric values (SERS intensity ratio) can effectively indicate the state of the molecules and avoid the above influence. Fig. 2C is the ratiometric values of characterized Raman peaks of the two SERS probes of the SERS- active microneedles, which had lower variabilities than that of their SERS intensity. Thus, the SERS intensity ratios of characterized Raman peaks, I1143 cm-1/I1180 cm-1 and I1606 cm-1/I1666 cm-1, were used as indicators of pH values and redox potentials of their environment.


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Figure 2. (A) SERS spectra of pH-sensitive and redox-sensitive SERS probes of a multiplexed SERS-active microneedle (the intensity of peak labeled by a grey area was use in Fig. 2B, and the intensity ratio of peaks labeled by a dash line area was use in Fig. 2C ), (B) SERS intensities of characterized Raman peaks at 1666 cm-1 of redox-sensitive SERS probes and 1085 cm-1 of pH-sensitive SERS probes, respectively, and (C) SERS intensity ratios of I1143 cm-1/I1180 cm-1 of pH-sensitive SERS probes and I1606 cm-1/I1666 cm-1 of redox-sensitive SERS probes, respectively. Fig. 3 showed the plots of the ratiometric values of the two SERS probes to pH values after the SERS-active microneedles were immersed in buffers with different pH values. The results demonstrated that the two SERS probes loaded in grooves still responded to their environment. As shown in Fig. 3B, the ratiometric values of the redox-sensitive SERS probes decreased with the increased pH value. In the next experiments, the ratiometric values of the redox-sensitive SERS probes were adjusted according to Fig. 3B.


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Figure 3. The response of multiplexed SERS-active microneedles in buffers with different pH values: (A) A plot of SERSI1143 cm-1/SERSI1180 cm-1 versus pH and (B) A plot of SERSI1606 cm-1 I1666 cm versus

1/SERS

pH.

Redox and pH response of multiplexed SERS-active microneedles in vivo. To assess their response in vivo, the SERS-active microneedles were inserted into a muscle of the hindleg of rats. As shown in Fig. 4, the SERS-active microneedles responded the environment of the muscle, and the two ratiometric values were up to reach a plateau in 5 min, which proved that the SERS-active microneedles can rapidly sense the environment (pH and redox potential) in vivo.

Figure 4. The response of multiplexed SERS-active microneedles in muscles for different time: (A) A plot of SERSI1143 cm-1/SERSI1180 cm-1 versus time and (B) A plot of SERSI1606 cm1/SERS

-1 I1666 cm versus

time.

To assess their response in vivo further, the solutions of lactic acid and N-acetyl-L-cysteine were injected into the muscle of the hindlegs respectively and then, SERS-active microneedles were inserted into the muscle to sense the change in pH and redox potential. Fig. 5A and 5B showed the SERS spectra of SERS-active microneedles in 0.9% saline, 20 g/L of lactic acid solution containing 0.9% of NaCl, and 10 g/L N-acetyl-L-cysteine solution containing 0.9% of NaCl, respectively. In saline, the ratiometric values from pH-sensitive and redox-sensitive SERS


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probes were 0.42 and 0.37, respectively; in lactic acid solution, the ratiometric value from pHsensitive SERS probes indicated that the solution was acidic, and its pH value was 4.0, and the adjusted ratiometric value of the redox-sensitive microneedles was 0.35, near the value in saline; in the N-acetyl-L-cysteine solution, the ratiometric value from pH-sensitive SERS probes indicated that the solution was also acidic, and its pH value was lower than 4.0, and the adjusted ratiometric value of the redox-sensitive microneedles was 0.50, higher than the value in saline, which suggested that the solution was reductive due to the reductivity of N-acetyl-L-cysteine.

Figure 5. SERS spectra of pH-sensitive SERS probes (A) and redox-sensitive SERS probes (B) of SERS-active microneedles in saline, lactic acid, and N-acetyl-L-cysteine solutions, respectively (the insets show the magnified spectra of characterized Raman peaks (labeled by dash lines) of ratiometric values), and the evolution of pH (C) and redox potential (D) in muscles after injection of lactic acid, and N-acetyl-L-cysteine, respectively.


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After injection of lactic acid and N-acetyl-L-cysteine, the environment evolution of the muscles was shown in Fig. 5C and 5D. After injection of lactic acid, the pH value in muscles decreased down to the valley bottom at 10 min and then, increased and recovered to physiological pH value at 40 min. Meanwhile, the ratiometric values of redox-sensitive SERS probes changed as shown in Fig. 4B, which suggested that the injection of lactic acid only changed the pH value, not the redox of the muscle. After injection of N-acetyl-L-cysteine, the pH values decreased sharply down to the bottom in 10 min, increased higher than the physiological pH value at 20 min, and then, decreased and recovered to physiological pH value at 40 min. Meanwhile, the ratiometric values of redox-sensitive SERS probes were higher than physiological value at 10 and 20 min, and recovered to physiological value at 40 min, which suggested that the injection of N-acetyl-L-cysteine not only changed the pH value but also changed the redox of the muscle. Besides, the reason of the difference of pH evolution in Fig. 5C may be that N-acetyl-L-cysteine or cysteine changed the signal pathways of cells in the muscles, and that lactic acid was only transported away from the muscles.24,25 To the best of our knowledge, no similar results of the evolution of redox and pH in muscles have been reported. Detection of redox potential and pH in rat joints simultaneously. Arthritis becomes more common with age, inducing the loss of function and the deformation of body. Up to now, there is still no known cure for either rheumatoid or osteoarthritis. It is better to develop an analytical method for direct detecting biomarkers in joints not in blood. Here, an acupuncture needle with 260 μm of diameter, less than the size of common syringe needles, were utilized a carrier to fabricate a multiplexed SERS-active microneedle to inserted into rat joints to detect pH and redox status. As shown in Fig. 6, the ratiometric values from normal joints were 0.42 and 0.43, near that in muscles, and the values from arthritis model joints were not only statistically


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different from that of normal ones but also statistically different each other, which suggested that the pH values in arthritis model joints were higher than 7.4 and the redox potentials were lower than physiological redox potential (suggested that redox status in the arthritis model joints is more reductive than that in normal joints). The damage of cartilage was considered to be irreversible, but according to our previous report,22 in which appeared reductive status indicated that new skin was forming during the wound healing process, we speculated that when the injected sodium iodoacetate were metabolized or transported out the inflamed joints would be in self-recover process.

Figure 6. The ratiometric values of two SERS probes of multiplexed SERS-active microneedles sensing in normal and model rat joints (* P

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