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Splicing Nanoparticles-based ‘Click’ SERS Could Aid Multiplex Liquid Biopsy and Accurate Cellular Imaging Yi Zeng, Jia-Qiang Ren, Ai-Guo Shen, and Jiming Hu J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b04892 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 5, 2018
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Splicing Nanoparticles-based ‘Click’ SERS Could Aid Multiplex Liquid Biopsy and Accurate Cellular Imaging Yi Zeng†, Jia-Qiang Ren‡, Ai-Guo Shen†*, Ji-Ming Hu† †
Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China ‡
National & Local Joint Engineering Research Center for High-throughput Drug Screening Technology, Hubei Province Key Laboratory of Biotechnology of Chinese Traditional Medicine, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei University, Wuhan 430062, China Supporting Information Placeholder ABSTRACT: Here, a completely new readout technique, so called ‘Click’ SERS, has been developed based on Raman scattered light splice derived from nanoparticle (NP) assemblies. The single and narrow (1-2 nm) emission originating from triple bondcontaining reporters undergoes dynamic combinatorial output, by means of controllable splice of SERS-active NPs analogous to small molecule units in click chemistry. Entirely different to conventional ‘sole code related to sole target’ readout protocol, the intuitional, predictable and uniquely identifiable ‘Click’ SERS is relies on the number rather than the intensity of combinatorial emissions. By this technique, 10-plex synchronous biomarkers detection under a single scan, and accurate cellular imaging under double exposure have been achieved. ‘Click’ SERS demonstrated multiple single band Raman scattering could be an authentic optical analysis method in biomedicine.
Multiplex assays are powerful and pragmatic for clinical applications, 1, 2 such as real time molecular profiling in liquid biopsies and non-invasive characterization of circulating tumor cells (CTCs) to provide fruitful information such as tumor size and location.3, 4 As information provided by a single biomarker is insufficient, the use of multiplex biomarkers panels could improve the clinical value.5, 6 The conventional way is using a unique code from a particular reporter to identify a specific target, but the resolution of acquired information is restricted by the numbers of distinguishable codes. 7 The current optical readouts, e.g., colorimetry, 8 fluorescence, 9 and even surface enhanced Raman spectroscopy/scattering (SERS), 10 are suffering from spectra overlapping upon multiple-label assay. In special, SERS generates the narrowest line widths (~1 nm) over the other methods, typically like fluorescence (~50 nm). In contrast, the fingerprint feature of SERS still leads to invalid quantification output considering the overlapping Raman bands. 11-12 Despite effort in multiplex assay development, the routine regarding ‘sole code related to sole target’ readout still largely limited the multi-assay for no more than single digits. SERS also hold promising potential for tumor cellular imaging owing to the advantages of multiplexing, high photo stability and sensitivity. 13- 15 However, several drawbacks cannot be ignored. Firstly, the size of the nanoparticles (NPs) utilized as enhancing substrates is generally designed larger than the tumor cell mem-
brane receptor 16-17, leading to inaccurate cellular imaging. Secondly, the gravity of metal substrates causes nonspecific adsorption, producing false positive results.18 Thirdly, during inevitable long-time incubation, endocytosis of surface-attached NPs into the cell randomly has a serious adverse impact on accuracy. To solve both issues, ‘Click’ SERS, a completely new readout technique, has been developed based on Raman scattered light splice derived from NPs assemblies. Figuratively speaking, Click SERS is tentatively defined according to its similarity to click chemistry, which is not a single specific reaction, but describes a way of generating products by efficiently joining small ‘modular units’ (Figure 1). Under high thermodynamic driving force, Click reactions occur in one pot, are not disturbed by water, generate minimal and inoffensive by-products. Analogously, the triple bond containing reporters endow SERS-active NPs with single and narrow (1-2 nm) emission of in Raman silent region (ca. 1800-2600 cm-1),19-20 which are extremely suitable to be served as a typical ‘SERS unit’ for freewill peaks combination in Click SERS. Basically, the inter-particles plasmonic coupling makes the controllable and artificial splicing of the triple bond-tagged SERS-active NPs (or SERS units) highly possible for emitting dynamic combinatorial signal output of single peaks. The ‘switch on’ of Click SERS is flexibly triggered by general driving forces from chemical, biological and physical origins. Entirely different to conventional ‘sole code related to sole target’ readout protocol, Click SERS relies on the number and positions rather than the intensity of combinatorial emissions, which is intuitional, predictable and uniquely identifiable. Since the emission codes of Click SERS is obtained through combinatorial math, the multiplexing or coding capacity can be continuously increased as long as more distinguishable emission are employed. Thus, Click SERS is demonstrated for the first time on multiplex liquid biopsy based on one-step DNA hybridization reaction and logic computation, thus enables 10-plex biomarkers detection synchronously under one scan. Besides, by subtle design of Click SERS logic gate for cellular imaging, a special double exposure operation is initiated to technically reduce the size of membrane targeting probes by a large margin, which has largely decreased unspecific absorption and improves the accuracy. The success of these strategies demonstrated single bands Raman scattering could be an authentic optical analysis method in biomedicine far more than a powerful tool in routine molecular structure characterization.
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Herein, we synthesized a series of triple bond containing SERS reporter molecules with highly differential scattering section within ca. 1800-2600 cm-1, with sharp distinguishable peaks (Figure S1 and Table S1).21-22 Au NPs with a diameter of 20 nm were employed as enhancing substrate, enabling fairly strong SERS effect but ensuring high stability. With rationally designed polyadenine (polyA)-DNA, the capture DNA were modified on the triple bond-labelled Au NPs, guaranteeing one strand per NP.23 Upon input DNA addition, controllable splicing of SERS active NPs was further allowed by dimer formation depending on DNA hybridization. The inter-particle hot spots were formed, generating two target-specific combinatorial peaks, as the typical double code Click spectra output.
With specific number and position of emission for each target, the specificity of Click SERS was exhibited by each target-specific output mode, furthermore, the logic computation from our barcode assay could output one and only operation result for each target, thus highlighting the multiplex detection suitability of Click SERS.
Figure 2. (A) Scheme of 15 well plate for 10-plex DNA detection; (B) Top view of the detection wells with full spectra when 10 targets added; (C) TEM images of dimer formation; (D) The acquired Click spectra presented in the wells during sample 1 and 2 detection; (E) QR code system of the China online payment app ‘Alipay’; (F) Our direct and quick QR code of sample 1: DNA g, h, i, and 2: DNA a, d, f. Figure 1. Schematic demonstration of the concept of ‘Click SERS’ analogous to click chemistry. As a proof-of-concept clinical demonstration, we employed this strategy for 10-plex target DNA detection synchronously (see Table S2 for all DNA sequences a-i). Four batches of Au NPs were functionalized with different reporters and specific capture DNA, aiming for DNA detection by hybridization (Figure S1). And a 15-well plate shown in Figure 2A was used for spatial separation of probes. Herein, with four distinguishable single peaks, we set 10 detection cells consisting of 4 single- and 6 double-code Click spectra, with each corresponding to a target. Those 4 single code resulted from DNA hybridization between the Au NPs functionalized with identical reporters, and the 6 double-code reflected hybridization between different tags. Meanwhile, by specific combination of probes, 5 more wells were set for overall simultaneously 10-plex analysis, including 4 triple and 1 quadruple code. After targets introduced, efficient reaction could be demonstrated by the TEM images in Figure 2C and Figure S2. Dimers were generated with a yield of about 60%. In presence of all 10 targets, as shown in Figure 2B, specific Click spectra output is produced in every well (Figure S3). Through combination of single narrow peaks, each unique and distinguishable output Click spectrum consisted of double-, triple-, and quadruple-codes.
This multiplex liquid biopsy analysis system was based on a double combination procedure which consisted of initial one-step DNA hybridization reaction and then logic computation of result from Click spectra readout. Target containing solution was added to each well, and information from every compartment was collected. If the spectra matched its expected output code exactly, we considered it as a signal ‘ON’, displayed as yellow in the scan code in Figure 2F, or a ‘OFF’ as white. Here, we incorporated barcode and quick response code (QR code) together for multiplex target analysis. The integration of overall information manifested as a QR code similarly used in the popular ‘Alipay’ (Figure 2E), a third party online payment solution that is rapid, convenient and accurate by a mobile QR code. Essentially, under a single laser scan, the complete data directly displayed as a QR code for immediate, direct and accurate 10-plex target analysis synchronously. For example, sample 1 and sample 2 contained DNA g, h, i and a, d, f respectively. A four-input ‘Q AND T AND D AND S’ gate was constructed with the truth table in shown in Table S3. This coding system is potentially attractive for obtaining multiplex biomarker signatures in liquid biopsies for prompt clinical decisions. To further testify the practical application of Click spectra, a simultaneous detection of thrombin, ATP, and Hg2+ was also carried out (Figure S4&5).
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Journal of the American Chemical Society Click SERS for accurate cellular imaging was demonstrated by double exposure, and the specific design and rationale were clearly clarified in Supporting Information with scheme shown as Figure S6. Application was realized on profiling of over expressing receptors on the surface of Hela cells, and epidermal growth factor receptor (EGFR) was selected as a typical biomarker.18 First of all, a series of control experiments were proposed to reveal the influence of false positive effects from endocytosis and unspecific adsorption. Probes functionalized with EGFR aptamer and bare Au NPs were separately incubated with the cells for comparison, meanwhile, Au NPs with different sizes were used in both conditions to demonstrate the effect of unspecific adsorption and gravity. (See in Figure S7&8) As a result, there was still signal response by the probes without aptamer by both 60 and 20 nm Au NPs. And as predicted, the side effect of large Au NPs was obviously more severe than the smaller ones. With the generally utilized enhancing substrates (50-80 nm), it would be inaccurate using them to depict the locus of membrane receptors (12-20 nm). Thus 20 nm Au NPs utilized here reduced the unspecific absorption to a large extent. To isolate false positive signals from endocytosis, the asmentioned double exposure experiments were led out (Figure 3). Targeting probes tagged with OPE1 exhibited intense Raman signal at 2152 cm-1, depicting the rough morphology of cells. The following introduced gating probes tagged with MBN showing Raman signal in 2227 cm-1 indicated where probe 1 was still on the surface of cell after incubation. As a result, the yellow region in Figure 3 thus highlighted the real location of receptors, by the Click spectra consisting of dual code peaks on 2152 cm-1 and 2227 cm-1.
Figure 4. (A) SERS imaging of Hela cells incubated with the Click SERS probes for targeting and gating. Scale bar for 5 µm. The SERS spectra of point 1 with OPE1 (B), point 2 with MBN (C) and point 3 (D) show Click spectra of double codes. In conclusion, we have introduced a new concept of readout by dynamic spectra splicing, ‘Click SERS spectra’. Click spectra possess huge encoding capacity for both high-throughput multiple biomarkers identification and accurate cellular imaging. By circumventing the limitation of spectra overlapping, following ongoing progress in both multiplex liquid biopsy analysis and accurate molecular profiling of CTC heterogeneity, Click SERS may play a useful role in precise cancer diagnosis and treatment, revealing the potential of unlimited multiplex code and provides important insight into accurate imaging of single tumour cells.
ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Mechanism clarification, experimental section, DNA sequences, scheme illustration, and cell imaging.
AUTHOR INFORMATION Corresponding Author Prof. Dr. Ai-Guo Shen,
[email protected] Notes The authors declare no competing financial interest.
ACKNOWLEDGMENT
Figure 3. (A) SERS imaging of Hela cells incubated with the Click SERS probes for targeting and gating. Scale bar for 5 µm. The SERS spectra of point 1 (B), and point 2 (C) showing Click spectra of double codes. For unspecific adsorption, two targeting probes were both functionalized with EGFR aptamers but with different Raman reporters, and incubated with the cells at the same time. Owing to the double check, the location with both signal from OPE 1 and MBN were shown demonstrated the true receptors presences, namely the yellow region in Figure 4. The remaining red and green areas were actually false positive results caused by gravity.
This work is supported by National Natural Science Foundation of China (Nos. 21475100, 81471696, and 21775114). We express our sincere thanks to Kevin M. Koo from the University of Queensland for his general help in manuscript revision.
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