Biosensors
On-line SERS Detection of Single Bacterium Using Novel SERS Nanoprobes and A Microfluidic Dielectrophoresis Device Hsing-Ying Lin, Chen-Han Huang, Wen-Hsin Hsieh, Ling-Hsuan Liu, Yuan-Chuen Lin, Chia-Chun Chu, Shi-Ting Wang, I-Ting Kuo, Lai-Kwan Chau,* and Chiou-Ying Yang*
The integration of novel surface-enhanced Raman scattering (SERS) nanoprobes and a microfluidic dielectrophoresis (DEP) device is developed for rapid on-line SERS detection of Salmonella enterica serotype Choleraesuis and Neisseria lactamica. The SERS nanoprobes are prepared by immobilization of specific antibody onto the surface of nanoaggregate-embedded beads (NAEBs), which are silica-coated, dye-induced aggregates of a small number of gold nanoparticles (AuNPs). Each NAEB gives highly enhanced Raman signals owing to the presence of well-defined plasmonic hot spots at junctions between AuNPs. Herein, the on-line SERS detection and accurate identification of suspended bacteria with a detection capability down to a single bacterium has been realized by the NAEB−DEP−Raman spectroscopy biosensing strategy. The practical detection limit with a measurement time of 10 min is estimated to be 70 CFU mL−1. In comparison with whole-cell enzyme-linked immunosorbent assay (ELISA), the SERS-nanoprobe-based biosensing method provides advantages of higher sensitivity and requiring lower amount of antibody in the assay (100-fold less). The total assay time including sample pretreatment is less than 2 h. Hence, this sensing strategy is promising for faster and effective on-line multiplex detection of single pathogenic bacterium by using different bioconjugated SERS nanoprobes.
1. Introduction Rapid, effective, and reliable microbial detection and inspection with high sensitivity and selectivity is an important
Dr. H.-Y. Lin, Dr. C.-H. Huang, L.-H. Liu, S.-T. Wang, I.-T. Kuo, Prof. L.-K. Chau Department of Chemistry and Biochemistry and Center for Nano Bio-Detection (AIM-HI) National Chung Cheng University Chiayi 62102, Taiwan E-mail: [email protected] DOI: 10.1002/smll.201401526 small 2014, DOI: 10.1002/smll.201401526
and challenging issue in the fields of clinical diagnosis, pharmaceutical, food and water safety.[1] For the diagnosis of acute bacterial infection, initial antibiotic therapy, and food quality assurance, the identification of pathogens is essential
Prof. W.-H. Hsieh Department of Mechanical Engineering National Chung Cheng University Chiayi 62102, Taiwan Dr. Y.-C. Lin, C.-C. Chu, Prof. C.-Y. Yang Institute of Molecular Biology National Chung Hsing University Taichung 40227, Taiwan E-mail: [email protected]
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for the decision of subsequent treatment steps and making a timely response to possible risks. The conventional approach for pathogenic bacteria analysis is based on the isolation and enrichment in special growth media to obtain pure cultures, then followed by biochemical screening tests. While reliable, the processes tend to be tedious, labor-intensive, and time-consuming, especially for the slow-growing microorganisms. Thus, faster on-line identification techniques are urgently required. Many methods have been developed to shorten the detection and identification time in microbial detection, such as mass spectrometry,[2,3] fluorescence spectroscopy,[4–6] enzyme-linked immunosorbent assay (ELISA),[7] surface plasmon resonance (SPR) sensors,[8,9] piezoelectric biosensors,[10] and polymerase chain reaction (PCR),[4,11–13] etc. Among these techniques, the biosensor-based methods have reduced the detection time in the range of tens minute to several hours with detection limits typically varying from 102 to 107 CFU mL−1.[4,14,15] However, detection of a few pathogens, e.g., 0, the particle is attracted toward the electrodes, where the electric field maximizes. This is called positive DEP. If Re[fCM] < 0, a repelling force from the electrodes pushes the particles toward the central region, where the electric field minimizes. This is called negative DEP. In this study, the negative DEP combined with the microfluidic device is used to trap single bacterium from dilute suspensions within the microchannel at the predetermined location for high-quality Raman spectroscopy identification. Figure 4(a) exhibits the microfluidic DEP chip. The chip with a distance of 20 µm between opposite microelectrodes is employed in all subsequent measurements, as shown in Figure 4(b). The nonuniform electrical field for negative DEP is generated by applying an alternating voltage of about 10 V to the microelectrodes via wire connections (Figure 4(d)). The adjacent electrodes are arranged to have opposite potential. Figure 4(c) shows the numerical simulation result of electric field distribution within the white square marked area in Figure 4(b). The simulation was performed using the commercial software CFDRCACE+ (ESI Group, France). The microelectrode tips are with higher electric field intensity up to 5 × 1011 V2 m−2. The intensity gradients drop down to a minimum value at the central region pointed by the four microelectrodes. The resultant negative DEP forces point toward the center between the four microelectrodes for efficient capture of the flowing suspended particle, as shown in the insets of Figure 5.
2.3. Multiplex Detection of Single Mimic Bacterium To demonstrate the feasibility of single bacterial detection with the combination of SERS nanoprobes and microfluidic small 2014, DOI: 10.1002/smll.201401526
Figure 5. SERS spectra of a DEP-captured PS microsphere as a mimic bacterium with a size of (a) 2.0 µm, (b) 6.0 µm, and (c) 10.0 µm, whose ligand-conjugated surface was recognized by different SERS nanoprobes. (d) The reference Raman spectrum of a DEP-captured PS microsphere with a size of 4.5 µm without surface modification. Insets display the corresponding microscopic images of the DEP-captured PS microsphere. The scale bar is 20 µm.
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DEP device, the ligand-conjugated PS microspheres in three distinct sizes acting as mimic bacteria are mixed with three kinds of SERS nanoprobes for the tests of on-line multiplex SERS detection. The PS microspheres in sizes of 2.0, 6.0, and 10.0 µm were separately modified with DNP, OVA, and biotin. The MGITC-, DTDCI-, and TRITC-labeled NAEBs were functionalized with a bioreceptor, anti-DNP antibody, anti-OVA antibody, and streptavidin, respectively. After incubation for one hour at room temperature, a mixture solution including the three kinds of ligand-conjugated PS microspheres and the blank 4.5-µm PS microspheres was pumped into the microfluidic DEP device for Raman spectroscopy identification. The flow rate was 20 µL min−1. The frequency of the triggering sine wave for DEP was 110 kHz. The flowing suspended microparticle was randomly trapped by the negative DEP device. Figure 5 displays the average Raman identification results (at least ten spectra were collected from different microparticles in each size). The spectrum of Figure 5(a), (b), and (c) includes characteristic bands of both PS microsphere and Raman reporter in respective SERS nanoprobes. The shadow strip and stars denote the Raman bands of 1004 and 1034 cm−1 belonging to the PS microsphere, as indicated by the reference spectrum of Figure 5(d). The built-in charge-coupled device (CCD) for microscope was used to assist the identification of the size of the trapped microparticles, as shown in the insets of Figure 5. The images directly verify the feasibility of multiplex detection of single mimic bacterium via the adoption of multiple highly specific SERS nanoprobes.
2.4. Single Bacterial Detection of S. Choleraesuis and N. lactamica S. Choleraesuis can cause a range of illnesses, from vomiting and diarrhea to life-threatening diseases.[45] N. lactamica is generally considered non-pathogenic.[46] Herein, S. Choleraesuis and N. lactamica are selected as examples to evaluate the capability of on-line SERS detection of single bacterium via the joint approach to include highly sensitive SERS nanoprobes and the microfluidic DEP device. Specificity of antibody is a critical issue for preparing SERS nanoprobes with high selectivity. Figure 6 exhibits the results of wholecell ELISA. S. Choleraesuis and N. lactamica with various concentrations (2 × 106 ∼ 2 × 109 CFU mL−1) were detected with the buffer diluted monoclonal antibody (mAb) 4B2D and 4–7–3,[47] respectively. The general characteristics of bacteria and antibodies used in this study are shown in Table 1. Results indicate that mAb 4B2D has high specificity to S. Choleraesuis (Figure 6(a)) and no cross reaction to N. lactamica (Figure 6(b)). On the contrary, mAb 4–7–3 has high specificity to N. lactamica (Figure 6(b)) and no cross reaction to S. Choleraesuis (Figure 6(a)). For both bacteria, the lowest bacterial concentration that can be determined by ELISA with 103-fold diluted antibody is 2 × 107 CFU mL−1. The use of lower antibody concentration will lead to significantly poorer ELISA sensitivity. On the other hand, 104-fold diluted antibody was good enough to be used to prepare SERS nanoprobes for single bacterial detection (Figure 7).
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Figure 6. Whole-cell ELISA using mAb 4B2D and mAb 4–7–3 for detection of (a) S. Choleraesuis and (b) N. lactamica in pure culture. C0 denotes the original concentration of antibody.
The DTDCI- and TRITC-labeled NAEBs were conjugated with mAb 4B2D and 4–7–3, respectively. Bacteria with a concentration of 2 × 107 CFU mL−1 were incubated with SERS nanoprobes of 4B2D-DTDCI-NAEBs and 4–7–3-TRITCNAEBs separately for an hour at room temperature. Insets of Figure 7 show the microscopic images of DEP-captured single bacterium as indicated by the white arrow, and SEM images display the single bacterial recognition results via
Table 1. Bacteria strains and monoclonal antibodies used Bacterium Shape
Salmonella Choleraesuis
Neisseria lactamica
Gram negative. Motile, non-sporing rod. 0.7 ∼ 1.5 µm by 2.0 ∼ 5.0 µm in size.[48]
Gram negative. Nonmotile cocci, occurring singly but more often in pairs with adjacent sides flattened. Flagella absent. 0.6 ∼ 1.0 µm in diameter.[49]
OU7085
49142
mAb (ca. 150 kDa)
mAb 4B2Da)
mAb 4–7–3b)
Target
flagellin FljB
Lipoprotein Ag473 (NMB1468)
Strain
a)mAb
4B2D was obtained from a mouse immunized with the SC strain OU7085 kindly pro-
vided by Dr. C. Chu, Department of Microbiology, Immunology, and Biopharmaceuticals, National Chiayi University, Taiwan; b)mAb 4–7–3 was raised against a serogroup B Neisseria meningitidis and it cross-reacts with N. lactamica as well as Neisseria gonorrhoeae.[47]
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Fast On-line SERS Detection of Single Bacterium
the spectrum (2) of Figure 7(a). On the other hand, results of determining the mixture of N. lactamica and 4–7–3-TRITCNAEBs shows the spectral peaks of TRITC, as shown in the spectrum (3) of Figure 7(b). In contrast, only the background was present for the negative control investigation of the mixture of S. Choleraesuis and 4–7–3-TRITC-NAEBs, as shown in the spectrum (4) of Figure 7(b). These spectral results demonstrate the feasibility of single bacterial detection by adopting the combination of microfluidic DEP device and the highly sensitive SERS nanoprobes with suitable recognition antibody. Additionally, to optimize the required antibody concentration for single bacterial detection, the SERS nanoprobes were functionalized in antibody solutions of different dilution folds (10−3 ∼ 10−5× initial antibody concentration) and then separately mixed with corresponding bacteria solution at a concentration of 2 × 107 CFU mL−1. Figure 8 exhibits the
Figure 7. Mean Raman spectra (calculated from 10 separate Raman spectra, double standard deviation depicted as corona) of DEP-captured single bacterium. (a) 4B2D-DTDCI-NAEBs mixed with S. Choleraesuis (SC) and N. lactamica (NL). (b) 4–7–3-TRITC-NAEBs mixed with SC and NL. (1): mixture of SC and 4B2D-DTDCI-NAEBs. (2): mixture of NL and 4B2D-DTDCI-NAEBs. (3): mixture of NL and 4–7–3-TRITC-NAEBs. (4): mixture of SC and 4–7–3-TRITC-NAEBs. Insets (5) and (7) show the corresponding microscopic images of DEP-captured single bacterium (white arrow). The scale bar is 10 µm. Insets (6) and (8) display SEM images of SERS nanoprobe-labeled S. Choleraesuis and N. lactamica, respectively.
the antibody-antigen reaction between the highly specific SERS nanoprobes and their corresponding bacterium. After incubation, the mixtures were individually pumped into the microfluidic DEP device for Raman spectroscopy determination. The flow rate was 15 µL min−1. The frequency of the triggering sine wave for DEP was 660 kHz. Figure 7 exhibits the mean Raman spectra of DEP-captured single bacterium (at least ten spectra were collected from different captures). For the mixture of S. Choleraesuis and 4B2D-DTDCINAEBs, the collected spectra showed the peaks of DTDCI, as shown in the spectrum (1) of Figure 7(a). However, for the negative control mixture of N. lactamica and 4B2D-DTDCINAEBs, only the background signal was present, as shown in small 2014, DOI: 10.1002/smll.201401526
Figure 8. Mean SERS spectra of DEP-captured single bacterium. (a) 4B2D-DTDCI-NAEBs mixed with S. Choleraesuis. (b) 4–7–3-TRITC-NAEBs mixed with N. lactamica. The antibody used in conjugation with NAEBs was diluted (1) 105-fold, (2) 20000-fold, (3) 104-fold, (4) 2000-fold, and (5) 103-fold of the original antibody solution. C0 denotes the initial concentration of antibody. (c) Calibration curve of the SERS intensity at 1242 cm−1 versus logarithmic concentration of mAb 4B2D, with R2 of 99.5%. (d) Calibration curve of the SERS intensity at 1355 cm−1 versus logarithmic concentration of mAb 4–7–3, with R2 of 99.3%. Error bars are calculated from 10 independent measurements.
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results of SERS detection of single bacterium on the microfluidic DEP device. For both bacteria, results of the mean SERS spectra reveal that the spectral intensity rises with increasing antibody concentration. The SERS nanoprobes with 103-fold diluted antibody for detection of single bacterium result in an ultrahigh SERS signal, as shown by spectrum (5) in both Figure 8(a) and (b). However, this antibody concentration is barely enough for ELISA in identifying bacteria at a concentration of 2 × 107 CFU mL−1 (Figure 6). When using the corresponding SERS nanoprobes with 105-fold diluted antibody for single bacterial detection, the spectral peaks of the embedded DTDCI or TRITC are still easily identified, as shown by spectrum (1) in both Figure 8(a) and (b). Thus, the required antibody concentration for single bacterial detection by NAEB−DEP−Raman spectroscopy approach can be 100-fold less than that required by ELISA, probably due to the higher reaction efficiency in solution than at surface and the high sensitivity of the NAEBs. Insets of Figure 8(c) and (d) show that the measured SERS intensity of nanoprobes at a fixed bacteria concentration has a linear relationship with the logarithmic concentration of antibody used to functionalize the nanoprobes. Results will be helpful for the preparation of multiple highly sensitive antibody-conjugated NAEBs that are specific to corresponding bacteria. Since the NAEB-based SERS nanoprobes can provide extremely high SERS intensity upon excitation, even a single SERS nanoprobe will provide an identifiable Raman signal.[29] As such, a single bacterium with a reasonable number of NAEBs attached to it can have an easily identified SERS signal. Thus, in comparison with the ELISA method employing the first antibody for recognition and the secondary antibody for signal amplification, our NAEB−DEP− Raman spectroscopy approach uses the SERS nanoprobes for both recognition and signal amplification. To compare the sensitivity of the two approaches based on the same dilution fold of antibody, the lowest bacterial concentration that can be detected via ELISA is 2 × 107 CFU mL−1, while single bacterium detection is achievable by our NAEB−DEP−Raman spectroscopy approach. In practice, the detection limit of a biosensor is often limited by analyte transport[50] and/or the practical time scale for measurements, that is to say, it is important to consider the minimum detectable concentration for a given measurement time. Such a practical detection limit holds especially for a flow-through counting scheme, as in our case here. If we assume that ∼10 counting events are required for reliable detection and taking account of the sample flow rate (15 µL min−1) in the microfluidic channel and the time required for trapping a bacterium (∼1 s), obtaining the Raman spectrum (1 s), and releasing the bacterium (∼1 s), then a measurement time window of 10 min will give a practical detection limit of 70 CFU mL−1 with our present NAEB− DEP−Raman spectroscopy approach. This is an exceptional low detection limit among many known biosensors.[51]
3. Conclusion and Outlook This study has successfully demonstrated the faster SERS detection of single bacterium with high specificity and
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sensitivity in a microchannel by using the combination of specific antibody-conjugated NAEBs and a microfluidic DEP device. Experimental results showed that the antibody concentration required in the functionalization of the NAEBs for SERS detection of S. Choleraesuis and N. lactamica was 100-fold less than that needed by the whole-cell ELISA. The entire diagnosis time including sample incubation and single bacterium detection was less than 2 h. In addition, multiple Raman tags incorporated with different antibodies were successfully employed for producing various SERS nanoprobes and applied to the on-line multiplex SERS detection of mimic bacteria with various sizes. Consequently, through substituting the antibody which is specific to other microorganisms, this on-line multiplex barcode biosensing strategy has the potential to provide a powerful platform for faster diagnosis of a wide variety of pathogenic bacteria and tumor cells in biomedical and biotechnological fields, especially for identifying uncultivable bacteria present in a small number.
4. Experimental Section Materials: Chemical and biochemical reagents, hydrogen tetrachloroaurate trihydrate (99.9%, HAuCl4·3H2O, SHOWA), sodium citrate tribasic dihydrate (99.0%, SHOWA), tetraethyl orthosilicate (99.0%, TEOS, Fluka), sodium hydroxide (97%, NaOH, SHOWA), (3-mercaptopropyl)-trimethoxysilane (95%, MPTMS, ACROS), (3-Aminopropyl)triethoxysilane (99%, APTES, Aldrich), ammonium hydroxide (28.0–30.0 wt.% in H2O, NH4OH, Sigma), anhydrous ethanol (EtOH, TEDIA), acetone (99.9%, TEDIA), sodium chloride (99.0%, NaCl, Sigma-Aldrich), potassium chloride (99.0%, KCl, SHOWA), monopotassium phosphate (99.0%, KH2PO4, SHOWA), disodium hydrogen phosphate (99.0%, Na2HPO4, SHOWA), N-Hydroxysuccinimide (98%, NHS, Sigma), N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (99.0%, EDC, Sigma), ethanolamine (99.0%, Sigma), Tween20 (Wako), malachite green isothiocyanate (MGITC, InvitrogenTM), 3,3′-Diethylthiadicarbocyanine iodide (98%, DTDCI, Aldrich), tetramethylrhodamine-5-isothiocyanate (TRITC, Sigma), polybead carboxylate microspheres (sizes of 2.00, 4.50, 6.00, and 10.00 µm, Polysciences, Inc.), N-(2,4-dinitrophenyl)-6-aminohexanoic acid (95%, DNP, Sigma), anti-dinitrophenyl antibody (anti-DNP, SigmaAldrich), ovalbumin (OVA, Sigma), monoclonal anti-ovalbumin antibody (anti-OVA, Sigma), biotin (Sigma-Aldrich), streptavidin (Sigma-Aldrich), bovine serum albumin (BSA, Sigma), MICROPOSIT S1818 positive photoresist and MICROPOSIT 351 developer (Rohm and Haas Electronic Materials LLC), were used as received. Unless otherwise noted, all solutions were prepared with ultrapure Milli-Q water (18.2 MΩ cm−1) from a Millipore Milli-Q system. Phosphatebuffered saline (PBS, pH 7.4) solution was composed of 150 mM NaCl, 4 mM KCl, 8.1 mM Na2HPO4, and 1.47 mM KH2PO4. Solution of phosphate-buffered saline with Tween20 (PBST, pH 7.4) was PBS containing 0.05% Tween20. The conductivity of PBST was 150 µs cm−1. Preparation of NAEBs: The AuNPs were synthesized by the following procedures. A 50 mL of 0.01% HAuCl4 solution was prepared and then heated under reflux with stirring in a two-necked flask for about 15 min. Then, 1 mL of a 1% sodium citrate solution was added to allow the reduction of HAuCl4. The solution was
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Fast On-line SERS Detection of Single Bacterium
continuously heated under stirring for additional 10 min. Afterward, it was allowed to cool to room temperature to form the AuNP solution. The NAEBs were prepared by the following steps.[29] 1 mL of ultrapure water was added to 4 mL of the AuNP solution with an initial pH value of ∼5, and adjusted to pH 10 by a 0.1 M NaOH solution. Then, a ∼10−4 M Raman reporter solution (70 µL MGITC, 100 µL DTDCI, or 80 µL TRITC) was introduced to the AuNP solution under vigorous stirring and allowed to equilibrate for 15 min. The Raman reporters reduce the surface zeta potential of AuNPs and result in the formation of nanoaggregates. Later, a coupling reagent of 20 µL of 5 × 10−5 M MPTMS/EtOH was added to the nanoaggregate solution under stirring. The stirring was then stopped to allow equilibration for 15 min. The MPTMS was used to render the surface of nanoaggregates vitreophilic and to promote the rapid coverage of silica on the outer surface of the nanoaggregates. Formation of the protective silica shell was achieved by using a modified Stöber process.[29] Solutions, 16 mL of EtOH, 500 µL of NH4OH, and 150 µL of 47.5 mM TEOS/EtOH, were quickly added in sequence to the nanoaggregate solution with vigorous shaking for 30 min. Then, 150 µL of 47.5 mM TEOS/EtOH was added to the mixture under stirring for additional 30 min. These two procedures were repeated two times. In total, 600 µL of 47.5 mM TEOS/ EtOH solution was added. Next, the mixture was under shaking for 3 h. Upon hydrolysis and condensation of the TEOS, a silica shell is completely formed to encapsulate the nanoaggregates. Afterward, the NAEB colloidal solution was dispensed and centrifuged at 12000 rpm for 12 min. The supernatant was carefully decanted. The precipitated NAEBs were washed with EtOH and centrifuged three times. Finally, the NAEBs were redispersed in 1 mL of ultrapure water. The SERS signal of NAEBs is reproducible after one month storage if they are kept at room temperature and in dark. Preparation of SERS Nanoprobes: The surface of NAEBs was first aminated by adding 1 mL solution of 1.4 × 1012 mL−1 NAEBs and 1 µL of APTES to 10 mL of EtOH under stirring. After stirring for 12 h, the mixture was transferred into a two-necked flask and heated at 70 °C under stirring for 1 h. Later, the solution was allowed to cool to room temperature, dispensed, and centrifuged at 8000 rpm for 5 min. The supernatant was removed. The precipitated NAEBs were rinsed with EtOH and centrifuged three times, and then redispersed in 1 mL of PBS solution for subsequent bioconjugation. To conjugate NAEBs with antibody for detection of mimic bacteria (PS microspheres), 1 mL of amine-functionalized NAEBs was mixed with 100 µL of 5 × 10−8 M bioreceptor solution (anti-DNP antibody, anti-OVA antibody, or streptavidin as the probe) and 20 µL of EDC/ NHS (2.0 × 10−2 M/5.0 × 10−3 M) solution. For detection of S. Choleraesuis and N. lactamica, 1 mL of amine-functionalized NAEBs was mixed with 10 µL of monoclonal antibody diluted to 103∼105fold in PBS. The mixture was under stirring at room temperature for 2 h and then centrifuged at 8000 rpm for 5 min to remove excess reactants. To reduce the effect of nonspecific binding, the precipitates were treated with 1% (vol/vol) BSA in 1 mL of PBS buffer. After shaking for 30 min, the antibody-conjugated NAEBs were rinsed with PBS and centrifuged for three times, redispersed in 1 mL of PBST solution, and stored at 4 °C for usage. Surface Modification of Polystyrene (PS) Microspheres: Polybead carboxylate microspheres are monodisperse PS microspheres that contain surface carboxyl groups. To demonstrate the capability of real-time SERS sensing scheme, PS microspheres were employed as mimic bacteria via conjugation of a specific ligand on small 2014, DOI: 10.1002/smll.201401526
their surface. A 10 µL of ∼107 mL−1 PS microspheres was diluted by 1 mL of PBS buffer and mixed with 20 µL of EDC/NHS (2.0 × 10−2 M/5.0 × 10−3 M) solution. After stirring at room temperature for 2 h, the carboxylate-functionalized PS microspheres were rinsed three times to remove excess reagents with PBS buffer and centrifuged at 8000 rpm for 5 min. The precipitated PS microspheres were redispersed in 1 mL of PBS buffer, and subsequently mixed with 100 µL of 5 × 10−8 M ligand (DNP, OVA, or biotin) solution at room temperature. The PS microspheres of 2.0, 6.0, and 10.0 µm were separately mixed with DNP, OVA, and biotin, respectively. After stirring for 2 h, the unbound ligands were rinsed away with PBS buffer and centrifuged at 8000 rpm for 5 min three times. The unreacted carboxyl groups on the surface of the PS microspheres were deactivated by rinsing with an aqueous solution of 1 M ethanolamine at pH 8.5 for 30 min. Finally, the ligand-conjugated PS microspheres were rinsed thoroughly with PBS solution and centrifuged at 8000 rpm for 5 min three times, and redispersed in 1 mL of PBS buffer. Preparation of Bacterial Samples for Raman Detection: S. Choleraesuis (SC), N. lactamica (NL), and mAb 4B2D and mAb 4–7–3[47] against bacteria were prepared by Prof. Yang’s laboratory. Information about the monoclonal antibodies and bacteria is given in Table 1. Bacteria used in this study were heat-inactivated (56 °C, 30 min) for safety issue. A 500 µL of bacterial suspenstion with suitable dilutions was dispersed into a 500 µL of antibodyconjugated NAEBs in PBST buffer, vortexed, and allowed to react for 1 h at room temperature. To remove the free SERS nanoprobes that might not bind to bacteria, the sample mixtures were centrifuged at 3000 rpm for 5 min. The supernatant was then discarded. The precipitates were washed with PBST and centrifuged three times, redispersed in 1 mL of PBST buffer, and analyzed by the DEP−Raman spectroscopy. Whole-cell ELISA: Binding of antibodies to SC and NL was respectively determined by whole-cell ELISA according to previous studies with slight modifications.[47,52] SC were grown overnight on LB agar plates at 37 °C and NL were on chocolate agar plates in a humidified 37 °C, 5% CO2 incubator. Then, the bacteria were harvested from agar plates using a sterile cotton swab and resuspended in PBS. The cells were washed with PBS (pH 7.2), centrifuged (at 8000 rpm for SC and at 3000 rpm for NL) for 10 min, resuspended in PBS, and heat inactivated for 30 min at 56 °C. For the quantification of bacterial concentration, the optical density at 600 nm (OD600) of bacteria in PBS solution was measured by a spectrophotometer (ChromTech CT-2300) with PBS as the reference background. Specific bacterial concentrations were prepared in PBS via serial dilutions for subsequent ELISA. The wells of microtitre plates (COSTAR 96-Well Flat Bottom EIA/RIA plate, Cat. No.9018) were coated by adding 50 µL/well of the suspension with specific bacterial concentrations, and allowing them to evaporate overnight at 37 °C. Afterward, the wells were rinsed with 200 µL of PBS three times, added 100 µL of blocking buffer (2% skin milk in PBS), and incubated at 37 °C for 1 h. Next, the blocking buffer was decanted and the wells were washed with 200 µL of PBS three times. The monoclonal antibody was added (50 µl/well) and used at a working dilution in blocking buffer. After incubation for 2 h at room temperature, the wells were washed with 200 µL of PBS three times. Bound antibody was revealed by adding 50 µL/well of goat anti-mouse polyvalent Ig/AP conjugate (A0162, SigmaAldrich) diluted to 1:3000 in blocking buffer, rinsing with 200 µL of PBS three times, and adding 50 µL/well AP substrate buffer
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(1 mg mL−1 p-nitrophenyl phosphate (pNPP), 0.5 mM MgCl2, 1 M diethanolamine (pH 9.8)). After 1 h incubation in dark at room temperature, the optical density at 405 nm (OD405) was measured by an ELISA reader (Tecan Sunrise Microplate Reader). Preparation of the DEP Microfluidic Device: The negative DEP with a quadrupole-electrode arrangement where four microelectrodes pointed towards a central region was employed in this study. The microfluidic DEP chip was integrated by the microfluidic channel and microelectrodes. The microfluidic part was produced by an injection molding technique. The cyclic olefin copolymer (COC) top plate had a dimension of 4.0 cm in length, 4.0 cm in width, and 0.2 cm in thickness with an interior microfluidic channel having 4.0 cm in length, 800 µm in width, and 800 µm in depth. An inlet and an outlet on the top plate for sample introduction were connected to two small access holes and mechanically bored into the microfluidic channel. The microelectrodes were fabricated by a standard photolithography technique on a gold-coated glass substrate. Positive photoresist (S1818) was spin-coated on the sputtered metal layer (100-nm gold layer with 100-nm chromium base layer) and patterned by a photolithography technique with a defined microelectrode geometry. The reacted photoresist was removed via immersion in a diluted MP351 developer, and the exposed metal layer was then etched. After etching, the remaining photoresist was washed away with acetone, methanol, and ultrapure water, and dried by blowing nitrogen gas. The microfluidic top plate was bonded to the microelectrode-patterned glass substrate by a UV curable adhesive glue. Due to the lower viscosity and the surface tension of the glue, the glue filled the contact interface between the top plate and the substrate by capillary action but did not enter the channel. These plates were then exposed to UV light to cure the bonding of the two plates together. The microfluidic channel of the integrated microfluidic DEP chip was washed with ethanol and ultrapure water prior to use. System and Measurement: All of the Raman spectra were collected under ambient conditions. The Raman spectroscopic measurements were carried out with a confocal Raman microscopy (XploRA, HORIBA Jobin Yvon) that allows automated measurements with an excitation of 638 nm and 5 mW from a solid-state laser. The pinhole of 300 µm was used for confocality. After removal of the Rayleigh scattering via an edge filter, the backscattered Raman light was diffracted by a spectrometer with a 1200 lines/mm grating and collected via a thermoelectrically cooled CCD operated at −70 °C. The integration time per Raman spectrum (600 to 1800 cm−1) was one second. The recorded Raman spectrum for subsequent characterization was averaged from three individual spectra. The SERS spectra of NAEBs in a quartz cube were obtained with a 10× objective (OLYMPUS MPLFLN, N.A. = 0.3) and regenerated by normalization of each SERS spectrum to the background. The background was obtained by measuring the blank of ultrapure water in a quartz cube. Figure 4(d) exhibits the configuration of SERS detection of bacterial sample on a microfluidic DEP device. The sample and PBST buffer were respectively pumped through Teflon tubes via 1 mL syringes and mechanical microliter syringe pumps, allowing a steady flow of sample through the microchannel at a controllable flow rate ranging from 15∼20 µL min−1. A function generator (GFG-8255, Instek) was used to give a 10 V sinusoidal wave with an adjustable frequency of 100 kHz∼1 MHz to trigger the generation of non-contact DEP forces in the quadrupole-electrode area through the wire connections to four microelectrodes. By
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controlling the frequency and flow rate, the number of trapped bacteria in the quadrupole detection zone can be maintained to just one. SERS spectra of the captured single bacterium were obtained with a 50× objective (OLYMPUS MPLFLN, N.A. = 0.8). The reference background was measured under the same condition without any trapped bacterium in the detection zone. Corresponding microscopic images of sample detection were collected by a CCD. Characterization: The size and morphology of AuNPs and NAEBs were determined by a transmission electron microscopy (TEM, JEOL JEM-2010) operated at 200 keV. The surface morphology of PS microspheres and bacteria in conjugation with SERS nanoprobes were verified by a field-emission scanning electron microscopy (FE-SEM, Hitachi 4800I) operated at 15 keV. Absorption spectra of AuNP solution and NAEBs in ultrapure water were respectively measured by a UV-Vis-NIR spectrophotometer (Jasco V-570).
Acknowledgements H.-Y. Lin and C.-H. Huang contributed equally to this work. The study was financially supported by Ministry of Science and Technology, Taiwan (NSC 101–2627-M-194–002, NSC 102–2627-M-194–002, NSC 101–2627-M-005–005, and NSC 102–2627-M-005–002).
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On-line SERS Detection of Single Bacterium Using Novel SERS Nanoprobes and A Microfluidic Dielectrophoresis Device Hsing-Ying Lin, Chen-Han Huang, Wen-Hsin Hsieh, Ling-Hsuan Liu, Yuan-Chuen Lin, Chia-Chun Chu, Shi-Ting Wang, I-Ting Kuo, Lai-Kwan Chau,* and Chiou-Ying Yang*
The integration of novel surface-enhanced Raman scattering (SERS) nanoprobes and a microfluidic dielectrophoresis (DEP) device is developed for rapid on-line SERS detection of Salmonella enterica serotype Choleraesuis and Neisseria lactamica. The SERS nanoprobes are prepared by immobilization of specific antibody onto the surface of nanoaggregate-embedded beads (NAEBs), which are silica-coated, dye-induced aggregates of a small number of gold nanoparticles (AuNPs). Each NAEB gives highly enhanced Raman signals owing to the presence of well-defined plasmonic hot spots at junctions between AuNPs. Herein, the on-line SERS detection and accurate identification of suspended bacteria with a detection capability down to a single bacterium has been realized by the NAEB−DEP−Raman spectroscopy biosensing strategy. The practical detection limit with a measurement time of 10 min is estimated to be 70 CFU mL−1. In comparison with whole-cell enzyme-linked immunosorbent assay (ELISA), the SERS-nanoprobe-based biosensing method provides advantages of higher sensitivity and requiring lower amount of antibody in the assay (100-fold less). The total assay time including sample pretreatment is less than 2 h. Hence, this sensing strategy is promising for faster and effective on-line multiplex detection of single pathogenic bacterium by using different bioconjugated SERS nanoprobes.
1. Introduction Rapid, effective, and reliable microbial detection and inspection with high sensitivity and selectivity is an important
Dr. H.-Y. Lin, Dr. C.-H. Huang, L.-H. Liu, S.-T. Wang, I.-T. Kuo, Prof. L.-K. Chau Department of Chemistry and Biochemistry and Center for Nano Bio-Detection (AIM-HI) National Chung Cheng University Chiayi 62102, Taiwan E-mail: [email protected] DOI: 10.1002/smll.201401526 small 2014, DOI: 10.1002/smll.201401526
and challenging issue in the fields of clinical diagnosis, pharmaceutical, food and water safety.[1] For the diagnosis of acute bacterial infection, initial antibiotic therapy, and food quality assurance, the identification of pathogens is essential
Prof. W.-H. Hsieh Department of Mechanical Engineering National Chung Cheng University Chiayi 62102, Taiwan Dr. Y.-C. Lin, C.-C. Chu, Prof. C.-Y. Yang Institute of Molecular Biology National Chung Hsing University Taichung 40227, Taiwan E-mail: [email protected]
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for the decision of subsequent treatment steps and making a timely response to possible risks. The conventional approach for pathogenic bacteria analysis is based on the isolation and enrichment in special growth media to obtain pure cultures, then followed by biochemical screening tests. While reliable, the processes tend to be tedious, labor-intensive, and time-consuming, especially for the slow-growing microorganisms. Thus, faster on-line identification techniques are urgently required. Many methods have been developed to shorten the detection and identification time in microbial detection, such as mass spectrometry,[2,3] fluorescence spectroscopy,[4–6] enzyme-linked immunosorbent assay (ELISA),[7] surface plasmon resonance (SPR) sensors,[8,9] piezoelectric biosensors,[10] and polymerase chain reaction (PCR),[4,11–13] etc. Among these techniques, the biosensor-based methods have reduced the detection time in the range of tens minute to several hours with detection limits typically varying from 102 to 107 CFU mL−1.[4,14,15] However, detection of a few pathogens, e.g., 0, the particle is attracted toward the electrodes, where the electric field maximizes. This is called positive DEP. If Re[fCM] < 0, a repelling force from the electrodes pushes the particles toward the central region, where the electric field minimizes. This is called negative DEP. In this study, the negative DEP combined with the microfluidic device is used to trap single bacterium from dilute suspensions within the microchannel at the predetermined location for high-quality Raman spectroscopy identification. Figure 4(a) exhibits the microfluidic DEP chip. The chip with a distance of 20 µm between opposite microelectrodes is employed in all subsequent measurements, as shown in Figure 4(b). The nonuniform electrical field for negative DEP is generated by applying an alternating voltage of about 10 V to the microelectrodes via wire connections (Figure 4(d)). The adjacent electrodes are arranged to have opposite potential. Figure 4(c) shows the numerical simulation result of electric field distribution within the white square marked area in Figure 4(b). The simulation was performed using the commercial software CFDRCACE+ (ESI Group, France). The microelectrode tips are with higher electric field intensity up to 5 × 1011 V2 m−2. The intensity gradients drop down to a minimum value at the central region pointed by the four microelectrodes. The resultant negative DEP forces point toward the center between the four microelectrodes for efficient capture of the flowing suspended particle, as shown in the insets of Figure 5.
2.3. Multiplex Detection of Single Mimic Bacterium To demonstrate the feasibility of single bacterial detection with the combination of SERS nanoprobes and microfluidic small 2014, DOI: 10.1002/smll.201401526
Figure 5. SERS spectra of a DEP-captured PS microsphere as a mimic bacterium with a size of (a) 2.0 µm, (b) 6.0 µm, and (c) 10.0 µm, whose ligand-conjugated surface was recognized by different SERS nanoprobes. (d) The reference Raman spectrum of a DEP-captured PS microsphere with a size of 4.5 µm without surface modification. Insets display the corresponding microscopic images of the DEP-captured PS microsphere. The scale bar is 20 µm.
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DEP device, the ligand-conjugated PS microspheres in three distinct sizes acting as mimic bacteria are mixed with three kinds of SERS nanoprobes for the tests of on-line multiplex SERS detection. The PS microspheres in sizes of 2.0, 6.0, and 10.0 µm were separately modified with DNP, OVA, and biotin. The MGITC-, DTDCI-, and TRITC-labeled NAEBs were functionalized with a bioreceptor, anti-DNP antibody, anti-OVA antibody, and streptavidin, respectively. After incubation for one hour at room temperature, a mixture solution including the three kinds of ligand-conjugated PS microspheres and the blank 4.5-µm PS microspheres was pumped into the microfluidic DEP device for Raman spectroscopy identification. The flow rate was 20 µL min−1. The frequency of the triggering sine wave for DEP was 110 kHz. The flowing suspended microparticle was randomly trapped by the negative DEP device. Figure 5 displays the average Raman identification results (at least ten spectra were collected from different microparticles in each size). The spectrum of Figure 5(a), (b), and (c) includes characteristic bands of both PS microsphere and Raman reporter in respective SERS nanoprobes. The shadow strip and stars denote the Raman bands of 1004 and 1034 cm−1 belonging to the PS microsphere, as indicated by the reference spectrum of Figure 5(d). The built-in charge-coupled device (CCD) for microscope was used to assist the identification of the size of the trapped microparticles, as shown in the insets of Figure 5. The images directly verify the feasibility of multiplex detection of single mimic bacterium via the adoption of multiple highly specific SERS nanoprobes.
2.4. Single Bacterial Detection of S. Choleraesuis and N. lactamica S. Choleraesuis can cause a range of illnesses, from vomiting and diarrhea to life-threatening diseases.[45] N. lactamica is generally considered non-pathogenic.[46] Herein, S. Choleraesuis and N. lactamica are selected as examples to evaluate the capability of on-line SERS detection of single bacterium via the joint approach to include highly sensitive SERS nanoprobes and the microfluidic DEP device. Specificity of antibody is a critical issue for preparing SERS nanoprobes with high selectivity. Figure 6 exhibits the results of wholecell ELISA. S. Choleraesuis and N. lactamica with various concentrations (2 × 106 ∼ 2 × 109 CFU mL−1) were detected with the buffer diluted monoclonal antibody (mAb) 4B2D and 4–7–3,[47] respectively. The general characteristics of bacteria and antibodies used in this study are shown in Table 1. Results indicate that mAb 4B2D has high specificity to S. Choleraesuis (Figure 6(a)) and no cross reaction to N. lactamica (Figure 6(b)). On the contrary, mAb 4–7–3 has high specificity to N. lactamica (Figure 6(b)) and no cross reaction to S. Choleraesuis (Figure 6(a)). For both bacteria, the lowest bacterial concentration that can be determined by ELISA with 103-fold diluted antibody is 2 × 107 CFU mL−1. The use of lower antibody concentration will lead to significantly poorer ELISA sensitivity. On the other hand, 104-fold diluted antibody was good enough to be used to prepare SERS nanoprobes for single bacterial detection (Figure 7).
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Figure 6. Whole-cell ELISA using mAb 4B2D and mAb 4–7–3 for detection of (a) S. Choleraesuis and (b) N. lactamica in pure culture. C0 denotes the original concentration of antibody.
The DTDCI- and TRITC-labeled NAEBs were conjugated with mAb 4B2D and 4–7–3, respectively. Bacteria with a concentration of 2 × 107 CFU mL−1 were incubated with SERS nanoprobes of 4B2D-DTDCI-NAEBs and 4–7–3-TRITCNAEBs separately for an hour at room temperature. Insets of Figure 7 show the microscopic images of DEP-captured single bacterium as indicated by the white arrow, and SEM images display the single bacterial recognition results via
Table 1. Bacteria strains and monoclonal antibodies used Bacterium Shape
Salmonella Choleraesuis
Neisseria lactamica
Gram negative. Motile, non-sporing rod. 0.7 ∼ 1.5 µm by 2.0 ∼ 5.0 µm in size.[48]
Gram negative. Nonmotile cocci, occurring singly but more often in pairs with adjacent sides flattened. Flagella absent. 0.6 ∼ 1.0 µm in diameter.[49]
OU7085
49142
mAb (ca. 150 kDa)
mAb 4B2Da)
mAb 4–7–3b)
Target
flagellin FljB
Lipoprotein Ag473 (NMB1468)
Strain
a)mAb
4B2D was obtained from a mouse immunized with the SC strain OU7085 kindly pro-
vided by Dr. C. Chu, Department of Microbiology, Immunology, and Biopharmaceuticals, National Chiayi University, Taiwan; b)mAb 4–7–3 was raised against a serogroup B Neisseria meningitidis and it cross-reacts with N. lactamica as well as Neisseria gonorrhoeae.[47]
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the spectrum (2) of Figure 7(a). On the other hand, results of determining the mixture of N. lactamica and 4–7–3-TRITCNAEBs shows the spectral peaks of TRITC, as shown in the spectrum (3) of Figure 7(b). In contrast, only the background was present for the negative control investigation of the mixture of S. Choleraesuis and 4–7–3-TRITC-NAEBs, as shown in the spectrum (4) of Figure 7(b). These spectral results demonstrate the feasibility of single bacterial detection by adopting the combination of microfluidic DEP device and the highly sensitive SERS nanoprobes with suitable recognition antibody. Additionally, to optimize the required antibody concentration for single bacterial detection, the SERS nanoprobes were functionalized in antibody solutions of different dilution folds (10−3 ∼ 10−5× initial antibody concentration) and then separately mixed with corresponding bacteria solution at a concentration of 2 × 107 CFU mL−1. Figure 8 exhibits the
Figure 7. Mean Raman spectra (calculated from 10 separate Raman spectra, double standard deviation depicted as corona) of DEP-captured single bacterium. (a) 4B2D-DTDCI-NAEBs mixed with S. Choleraesuis (SC) and N. lactamica (NL). (b) 4–7–3-TRITC-NAEBs mixed with SC and NL. (1): mixture of SC and 4B2D-DTDCI-NAEBs. (2): mixture of NL and 4B2D-DTDCI-NAEBs. (3): mixture of NL and 4–7–3-TRITC-NAEBs. (4): mixture of SC and 4–7–3-TRITC-NAEBs. Insets (5) and (7) show the corresponding microscopic images of DEP-captured single bacterium (white arrow). The scale bar is 10 µm. Insets (6) and (8) display SEM images of SERS nanoprobe-labeled S. Choleraesuis and N. lactamica, respectively.
the antibody-antigen reaction between the highly specific SERS nanoprobes and their corresponding bacterium. After incubation, the mixtures were individually pumped into the microfluidic DEP device for Raman spectroscopy determination. The flow rate was 15 µL min−1. The frequency of the triggering sine wave for DEP was 660 kHz. Figure 7 exhibits the mean Raman spectra of DEP-captured single bacterium (at least ten spectra were collected from different captures). For the mixture of S. Choleraesuis and 4B2D-DTDCINAEBs, the collected spectra showed the peaks of DTDCI, as shown in the spectrum (1) of Figure 7(a). However, for the negative control mixture of N. lactamica and 4B2D-DTDCINAEBs, only the background signal was present, as shown in small 2014, DOI: 10.1002/smll.201401526
Figure 8. Mean SERS spectra of DEP-captured single bacterium. (a) 4B2D-DTDCI-NAEBs mixed with S. Choleraesuis. (b) 4–7–3-TRITC-NAEBs mixed with N. lactamica. The antibody used in conjugation with NAEBs was diluted (1) 105-fold, (2) 20000-fold, (3) 104-fold, (4) 2000-fold, and (5) 103-fold of the original antibody solution. C0 denotes the initial concentration of antibody. (c) Calibration curve of the SERS intensity at 1242 cm−1 versus logarithmic concentration of mAb 4B2D, with R2 of 99.5%. (d) Calibration curve of the SERS intensity at 1355 cm−1 versus logarithmic concentration of mAb 4–7–3, with R2 of 99.3%. Error bars are calculated from 10 independent measurements.
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results of SERS detection of single bacterium on the microfluidic DEP device. For both bacteria, results of the mean SERS spectra reveal that the spectral intensity rises with increasing antibody concentration. The SERS nanoprobes with 103-fold diluted antibody for detection of single bacterium result in an ultrahigh SERS signal, as shown by spectrum (5) in both Figure 8(a) and (b). However, this antibody concentration is barely enough for ELISA in identifying bacteria at a concentration of 2 × 107 CFU mL−1 (Figure 6). When using the corresponding SERS nanoprobes with 105-fold diluted antibody for single bacterial detection, the spectral peaks of the embedded DTDCI or TRITC are still easily identified, as shown by spectrum (1) in both Figure 8(a) and (b). Thus, the required antibody concentration for single bacterial detection by NAEB−DEP−Raman spectroscopy approach can be 100-fold less than that required by ELISA, probably due to the higher reaction efficiency in solution than at surface and the high sensitivity of the NAEBs. Insets of Figure 8(c) and (d) show that the measured SERS intensity of nanoprobes at a fixed bacteria concentration has a linear relationship with the logarithmic concentration of antibody used to functionalize the nanoprobes. Results will be helpful for the preparation of multiple highly sensitive antibody-conjugated NAEBs that are specific to corresponding bacteria. Since the NAEB-based SERS nanoprobes can provide extremely high SERS intensity upon excitation, even a single SERS nanoprobe will provide an identifiable Raman signal.[29] As such, a single bacterium with a reasonable number of NAEBs attached to it can have an easily identified SERS signal. Thus, in comparison with the ELISA method employing the first antibody for recognition and the secondary antibody for signal amplification, our NAEB−DEP− Raman spectroscopy approach uses the SERS nanoprobes for both recognition and signal amplification. To compare the sensitivity of the two approaches based on the same dilution fold of antibody, the lowest bacterial concentration that can be detected via ELISA is 2 × 107 CFU mL−1, while single bacterium detection is achievable by our NAEB−DEP−Raman spectroscopy approach. In practice, the detection limit of a biosensor is often limited by analyte transport[50] and/or the practical time scale for measurements, that is to say, it is important to consider the minimum detectable concentration for a given measurement time. Such a practical detection limit holds especially for a flow-through counting scheme, as in our case here. If we assume that ∼10 counting events are required for reliable detection and taking account of the sample flow rate (15 µL min−1) in the microfluidic channel and the time required for trapping a bacterium (∼1 s), obtaining the Raman spectrum (1 s), and releasing the bacterium (∼1 s), then a measurement time window of 10 min will give a practical detection limit of 70 CFU mL−1 with our present NAEB− DEP−Raman spectroscopy approach. This is an exceptional low detection limit among many known biosensors.[51]
3. Conclusion and Outlook This study has successfully demonstrated the faster SERS detection of single bacterium with high specificity and
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sensitivity in a microchannel by using the combination of specific antibody-conjugated NAEBs and a microfluidic DEP device. Experimental results showed that the antibody concentration required in the functionalization of the NAEBs for SERS detection of S. Choleraesuis and N. lactamica was 100-fold less than that needed by the whole-cell ELISA. The entire diagnosis time including sample incubation and single bacterium detection was less than 2 h. In addition, multiple Raman tags incorporated with different antibodies were successfully employed for producing various SERS nanoprobes and applied to the on-line multiplex SERS detection of mimic bacteria with various sizes. Consequently, through substituting the antibody which is specific to other microorganisms, this on-line multiplex barcode biosensing strategy has the potential to provide a powerful platform for faster diagnosis of a wide variety of pathogenic bacteria and tumor cells in biomedical and biotechnological fields, especially for identifying uncultivable bacteria present in a small number.
4. Experimental Section Materials: Chemical and biochemical reagents, hydrogen tetrachloroaurate trihydrate (99.9%, HAuCl4·3H2O, SHOWA), sodium citrate tribasic dihydrate (99.0%, SHOWA), tetraethyl orthosilicate (99.0%, TEOS, Fluka), sodium hydroxide (97%, NaOH, SHOWA), (3-mercaptopropyl)-trimethoxysilane (95%, MPTMS, ACROS), (3-Aminopropyl)triethoxysilane (99%, APTES, Aldrich), ammonium hydroxide (28.0–30.0 wt.% in H2O, NH4OH, Sigma), anhydrous ethanol (EtOH, TEDIA), acetone (99.9%, TEDIA), sodium chloride (99.0%, NaCl, Sigma-Aldrich), potassium chloride (99.0%, KCl, SHOWA), monopotassium phosphate (99.0%, KH2PO4, SHOWA), disodium hydrogen phosphate (99.0%, Na2HPO4, SHOWA), N-Hydroxysuccinimide (98%, NHS, Sigma), N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (99.0%, EDC, Sigma), ethanolamine (99.0%, Sigma), Tween20 (Wako), malachite green isothiocyanate (MGITC, InvitrogenTM), 3,3′-Diethylthiadicarbocyanine iodide (98%, DTDCI, Aldrich), tetramethylrhodamine-5-isothiocyanate (TRITC, Sigma), polybead carboxylate microspheres (sizes of 2.00, 4.50, 6.00, and 10.00 µm, Polysciences, Inc.), N-(2,4-dinitrophenyl)-6-aminohexanoic acid (95%, DNP, Sigma), anti-dinitrophenyl antibody (anti-DNP, SigmaAldrich), ovalbumin (OVA, Sigma), monoclonal anti-ovalbumin antibody (anti-OVA, Sigma), biotin (Sigma-Aldrich), streptavidin (Sigma-Aldrich), bovine serum albumin (BSA, Sigma), MICROPOSIT S1818 positive photoresist and MICROPOSIT 351 developer (Rohm and Haas Electronic Materials LLC), were used as received. Unless otherwise noted, all solutions were prepared with ultrapure Milli-Q water (18.2 MΩ cm−1) from a Millipore Milli-Q system. Phosphatebuffered saline (PBS, pH 7.4) solution was composed of 150 mM NaCl, 4 mM KCl, 8.1 mM Na2HPO4, and 1.47 mM KH2PO4. Solution of phosphate-buffered saline with Tween20 (PBST, pH 7.4) was PBS containing 0.05% Tween20. The conductivity of PBST was 150 µs cm−1. Preparation of NAEBs: The AuNPs were synthesized by the following procedures. A 50 mL of 0.01% HAuCl4 solution was prepared and then heated under reflux with stirring in a two-necked flask for about 15 min. Then, 1 mL of a 1% sodium citrate solution was added to allow the reduction of HAuCl4. The solution was
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continuously heated under stirring for additional 10 min. Afterward, it was allowed to cool to room temperature to form the AuNP solution. The NAEBs were prepared by the following steps.[29] 1 mL of ultrapure water was added to 4 mL of the AuNP solution with an initial pH value of ∼5, and adjusted to pH 10 by a 0.1 M NaOH solution. Then, a ∼10−4 M Raman reporter solution (70 µL MGITC, 100 µL DTDCI, or 80 µL TRITC) was introduced to the AuNP solution under vigorous stirring and allowed to equilibrate for 15 min. The Raman reporters reduce the surface zeta potential of AuNPs and result in the formation of nanoaggregates. Later, a coupling reagent of 20 µL of 5 × 10−5 M MPTMS/EtOH was added to the nanoaggregate solution under stirring. The stirring was then stopped to allow equilibration for 15 min. The MPTMS was used to render the surface of nanoaggregates vitreophilic and to promote the rapid coverage of silica on the outer surface of the nanoaggregates. Formation of the protective silica shell was achieved by using a modified Stöber process.[29] Solutions, 16 mL of EtOH, 500 µL of NH4OH, and 150 µL of 47.5 mM TEOS/EtOH, were quickly added in sequence to the nanoaggregate solution with vigorous shaking for 30 min. Then, 150 µL of 47.5 mM TEOS/EtOH was added to the mixture under stirring for additional 30 min. These two procedures were repeated two times. In total, 600 µL of 47.5 mM TEOS/ EtOH solution was added. Next, the mixture was under shaking for 3 h. Upon hydrolysis and condensation of the TEOS, a silica shell is completely formed to encapsulate the nanoaggregates. Afterward, the NAEB colloidal solution was dispensed and centrifuged at 12000 rpm for 12 min. The supernatant was carefully decanted. The precipitated NAEBs were washed with EtOH and centrifuged three times. Finally, the NAEBs were redispersed in 1 mL of ultrapure water. The SERS signal of NAEBs is reproducible after one month storage if they are kept at room temperature and in dark. Preparation of SERS Nanoprobes: The surface of NAEBs was first aminated by adding 1 mL solution of 1.4 × 1012 mL−1 NAEBs and 1 µL of APTES to 10 mL of EtOH under stirring. After stirring for 12 h, the mixture was transferred into a two-necked flask and heated at 70 °C under stirring for 1 h. Later, the solution was allowed to cool to room temperature, dispensed, and centrifuged at 8000 rpm for 5 min. The supernatant was removed. The precipitated NAEBs were rinsed with EtOH and centrifuged three times, and then redispersed in 1 mL of PBS solution for subsequent bioconjugation. To conjugate NAEBs with antibody for detection of mimic bacteria (PS microspheres), 1 mL of amine-functionalized NAEBs was mixed with 100 µL of 5 × 10−8 M bioreceptor solution (anti-DNP antibody, anti-OVA antibody, or streptavidin as the probe) and 20 µL of EDC/ NHS (2.0 × 10−2 M/5.0 × 10−3 M) solution. For detection of S. Choleraesuis and N. lactamica, 1 mL of amine-functionalized NAEBs was mixed with 10 µL of monoclonal antibody diluted to 103∼105fold in PBS. The mixture was under stirring at room temperature for 2 h and then centrifuged at 8000 rpm for 5 min to remove excess reactants. To reduce the effect of nonspecific binding, the precipitates were treated with 1% (vol/vol) BSA in 1 mL of PBS buffer. After shaking for 30 min, the antibody-conjugated NAEBs were rinsed with PBS and centrifuged for three times, redispersed in 1 mL of PBST solution, and stored at 4 °C for usage. Surface Modification of Polystyrene (PS) Microspheres: Polybead carboxylate microspheres are monodisperse PS microspheres that contain surface carboxyl groups. To demonstrate the capability of real-time SERS sensing scheme, PS microspheres were employed as mimic bacteria via conjugation of a specific ligand on small 2014, DOI: 10.1002/smll.201401526
their surface. A 10 µL of ∼107 mL−1 PS microspheres was diluted by 1 mL of PBS buffer and mixed with 20 µL of EDC/NHS (2.0 × 10−2 M/5.0 × 10−3 M) solution. After stirring at room temperature for 2 h, the carboxylate-functionalized PS microspheres were rinsed three times to remove excess reagents with PBS buffer and centrifuged at 8000 rpm for 5 min. The precipitated PS microspheres were redispersed in 1 mL of PBS buffer, and subsequently mixed with 100 µL of 5 × 10−8 M ligand (DNP, OVA, or biotin) solution at room temperature. The PS microspheres of 2.0, 6.0, and 10.0 µm were separately mixed with DNP, OVA, and biotin, respectively. After stirring for 2 h, the unbound ligands were rinsed away with PBS buffer and centrifuged at 8000 rpm for 5 min three times. The unreacted carboxyl groups on the surface of the PS microspheres were deactivated by rinsing with an aqueous solution of 1 M ethanolamine at pH 8.5 for 30 min. Finally, the ligand-conjugated PS microspheres were rinsed thoroughly with PBS solution and centrifuged at 8000 rpm for 5 min three times, and redispersed in 1 mL of PBS buffer. Preparation of Bacterial Samples for Raman Detection: S. Choleraesuis (SC), N. lactamica (NL), and mAb 4B2D and mAb 4–7–3[47] against bacteria were prepared by Prof. Yang’s laboratory. Information about the monoclonal antibodies and bacteria is given in Table 1. Bacteria used in this study were heat-inactivated (56 °C, 30 min) for safety issue. A 500 µL of bacterial suspenstion with suitable dilutions was dispersed into a 500 µL of antibodyconjugated NAEBs in PBST buffer, vortexed, and allowed to react for 1 h at room temperature. To remove the free SERS nanoprobes that might not bind to bacteria, the sample mixtures were centrifuged at 3000 rpm for 5 min. The supernatant was then discarded. The precipitates were washed with PBST and centrifuged three times, redispersed in 1 mL of PBST buffer, and analyzed by the DEP−Raman spectroscopy. Whole-cell ELISA: Binding of antibodies to SC and NL was respectively determined by whole-cell ELISA according to previous studies with slight modifications.[47,52] SC were grown overnight on LB agar plates at 37 °C and NL were on chocolate agar plates in a humidified 37 °C, 5% CO2 incubator. Then, the bacteria were harvested from agar plates using a sterile cotton swab and resuspended in PBS. The cells were washed with PBS (pH 7.2), centrifuged (at 8000 rpm for SC and at 3000 rpm for NL) for 10 min, resuspended in PBS, and heat inactivated for 30 min at 56 °C. For the quantification of bacterial concentration, the optical density at 600 nm (OD600) of bacteria in PBS solution was measured by a spectrophotometer (ChromTech CT-2300) with PBS as the reference background. Specific bacterial concentrations were prepared in PBS via serial dilutions for subsequent ELISA. The wells of microtitre plates (COSTAR 96-Well Flat Bottom EIA/RIA plate, Cat. No.9018) were coated by adding 50 µL/well of the suspension with specific bacterial concentrations, and allowing them to evaporate overnight at 37 °C. Afterward, the wells were rinsed with 200 µL of PBS three times, added 100 µL of blocking buffer (2% skin milk in PBS), and incubated at 37 °C for 1 h. Next, the blocking buffer was decanted and the wells were washed with 200 µL of PBS three times. The monoclonal antibody was added (50 µl/well) and used at a working dilution in blocking buffer. After incubation for 2 h at room temperature, the wells were washed with 200 µL of PBS three times. Bound antibody was revealed by adding 50 µL/well of goat anti-mouse polyvalent Ig/AP conjugate (A0162, SigmaAldrich) diluted to 1:3000 in blocking buffer, rinsing with 200 µL of PBS three times, and adding 50 µL/well AP substrate buffer
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(1 mg mL−1 p-nitrophenyl phosphate (pNPP), 0.5 mM MgCl2, 1 M diethanolamine (pH 9.8)). After 1 h incubation in dark at room temperature, the optical density at 405 nm (OD405) was measured by an ELISA reader (Tecan Sunrise Microplate Reader). Preparation of the DEP Microfluidic Device: The negative DEP with a quadrupole-electrode arrangement where four microelectrodes pointed towards a central region was employed in this study. The microfluidic DEP chip was integrated by the microfluidic channel and microelectrodes. The microfluidic part was produced by an injection molding technique. The cyclic olefin copolymer (COC) top plate had a dimension of 4.0 cm in length, 4.0 cm in width, and 0.2 cm in thickness with an interior microfluidic channel having 4.0 cm in length, 800 µm in width, and 800 µm in depth. An inlet and an outlet on the top plate for sample introduction were connected to two small access holes and mechanically bored into the microfluidic channel. The microelectrodes were fabricated by a standard photolithography technique on a gold-coated glass substrate. Positive photoresist (S1818) was spin-coated on the sputtered metal layer (100-nm gold layer with 100-nm chromium base layer) and patterned by a photolithography technique with a defined microelectrode geometry. The reacted photoresist was removed via immersion in a diluted MP351 developer, and the exposed metal layer was then etched. After etching, the remaining photoresist was washed away with acetone, methanol, and ultrapure water, and dried by blowing nitrogen gas. The microfluidic top plate was bonded to the microelectrode-patterned glass substrate by a UV curable adhesive glue. Due to the lower viscosity and the surface tension of the glue, the glue filled the contact interface between the top plate and the substrate by capillary action but did not enter the channel. These plates were then exposed to UV light to cure the bonding of the two plates together. The microfluidic channel of the integrated microfluidic DEP chip was washed with ethanol and ultrapure water prior to use. System and Measurement: All of the Raman spectra were collected under ambient conditions. The Raman spectroscopic measurements were carried out with a confocal Raman microscopy (XploRA, HORIBA Jobin Yvon) that allows automated measurements with an excitation of 638 nm and 5 mW from a solid-state laser. The pinhole of 300 µm was used for confocality. After removal of the Rayleigh scattering via an edge filter, the backscattered Raman light was diffracted by a spectrometer with a 1200 lines/mm grating and collected via a thermoelectrically cooled CCD operated at −70 °C. The integration time per Raman spectrum (600 to 1800 cm−1) was one second. The recorded Raman spectrum for subsequent characterization was averaged from three individual spectra. The SERS spectra of NAEBs in a quartz cube were obtained with a 10× objective (OLYMPUS MPLFLN, N.A. = 0.3) and regenerated by normalization of each SERS spectrum to the background. The background was obtained by measuring the blank of ultrapure water in a quartz cube. Figure 4(d) exhibits the configuration of SERS detection of bacterial sample on a microfluidic DEP device. The sample and PBST buffer were respectively pumped through Teflon tubes via 1 mL syringes and mechanical microliter syringe pumps, allowing a steady flow of sample through the microchannel at a controllable flow rate ranging from 15∼20 µL min−1. A function generator (GFG-8255, Instek) was used to give a 10 V sinusoidal wave with an adjustable frequency of 100 kHz∼1 MHz to trigger the generation of non-contact DEP forces in the quadrupole-electrode area through the wire connections to four microelectrodes. By
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controlling the frequency and flow rate, the number of trapped bacteria in the quadrupole detection zone can be maintained to just one. SERS spectra of the captured single bacterium were obtained with a 50× objective (OLYMPUS MPLFLN, N.A. = 0.8). The reference background was measured under the same condition without any trapped bacterium in the detection zone. Corresponding microscopic images of sample detection were collected by a CCD. Characterization: The size and morphology of AuNPs and NAEBs were determined by a transmission electron microscopy (TEM, JEOL JEM-2010) operated at 200 keV. The surface morphology of PS microspheres and bacteria in conjugation with SERS nanoprobes were verified by a field-emission scanning electron microscopy (FE-SEM, Hitachi 4800I) operated at 15 keV. Absorption spectra of AuNP solution and NAEBs in ultrapure water were respectively measured by a UV-Vis-NIR spectrophotometer (Jasco V-570).
Acknowledgements H.-Y. Lin and C.-H. Huang contributed equally to this work. The study was financially supported by Ministry of Science and Technology, Taiwan (NSC 101–2627-M-194–002, NSC 102–2627-M-194–002, NSC 101–2627-M-005–005, and NSC 102–2627-M-005–002).
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Fast On-line SERS Detection of Single Bacterium
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Received: May 29, 2014 Revised: July 12, 2014 Published online:
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