A method for improving performance of a SERS sensor by using a bifunctional small molecule to assist modification

Abstract

The application utilizes a method for improving the performance of a SERS sensor by means of a bifunctional small molecule assisted modification, provides a cell silencing zone peak for Raman detection, improves the detection sensitivity, and the small molecule has the function of a blocking agent, effectively reduces non-specific adsorption and uncontrollable aggregation, significantly improves the stability, provides the possibility for controllable aggregation in a complex environment in cells, and improves the detection accuracy. The method has simple preparation and convenient operation, the prepared sensor has high sensitivity and good accuracy, and even in the complex environment in cells, the sensor also has good monitoring of the pH change process generated by lysosome maturation, thereby providing a feasible method for diagnosis and early treatment of related diseases.

Description

Technical Field

[0001] This invention relates to a method for preparing a SERS sensor using bifunctional small molecule-assisted modification. This invention belongs to the field of nanomaterials and has potential applications in Raman enhancement and dynamic pH monitoring. Background Technology

[0002] Surface-enhanced Raman spectroscopy (SERS) technology boasts ultra-high sensitivity, enabling significant Raman signal amplification from plasmonic nanostructures and even single-molecule detection. Compared to most fluorescent probes, SERS tags are not easily photobleached, a significant advantage. More importantly, they allow the detection of many inactive or overly complex molecules.

[0003] With the continuous development of SERS technology, silent region Raman dyes have been used to detect Raman signals in complex biological samples. These molecules have a Raman signal range of 1800-2800 cm⁻¹. -1 The peaks emerge within a certain range and do not interfere with the Raman signal peaks of proteins, peptides, amino acids, and other substances present in the biological sample itself, which can further improve the sensitivity of Raman detection. It has been used in the construction of various Raman sensors.

[0004] In most cases, a single plasmonic nanoparticle cannot provide sufficient signal enhancement for practical applications. However, when two nanoparticles are brought close enough together, they can generate a significant signal enhancement within their gap. This spatial region exhibiting extremely high electric field enhancement and producing a strong SERS signal is called a hotspot, and hotspots typically exist within the gaps between nanoparticle aggregates. Theoretical calculations show that the enhancement factor of a silver dimer with a spacing of 2 nm is approximately 10 times that of a silver sphere. 4 Therefore, designing SERS substrates with effective coupling between nanostructures to provide the highest sensitivity is crucial for analytical detection.

[0005] i-motif is composed of four repeating cytosine sequences via C·CH + The i-motif, a four-stranded structure formed by base pairs, requires weakly acidic conditions for stability. This unique property makes it the first DNA molecular motor driven by pH changes. Studies have shown that it exhibits a millisecond-level response speed, approaching the allosteric rate of natural proteins. Moreover, the i-motif structure's response to different pH values ​​is reversible. More importantly, when DNA sequences fold into three-dimensional i-motif structures under appropriate pH conditions, this characteristic can be used to construct ideal spatial coupling effects.

[0006] Information about changes in endosomal pH over time plays a crucial role in many pathologies, and understanding endocytosis helps in comprehending nanobiological interactions and drug delivery. Although the lysosomal pathway is extremely important for many intracellular functions, there have been few previous reports on the application of SERS to study and characterize this process.

[0007] However, improving the stability and controllable aggregation of SERS sensors in complex environments remains a critical issue to be addressed. In summary, this invention aims to utilize Raman dyes in the cell silencing region to exert a dual-function effect, constructing an i-motif-based SERS sensor capable of dynamically monitoring changes in intracellular pH. This enables dynamic monitoring of pH in the endosome-lysosome process, improves the stability of the probe in complex environments, achieves controllable aggregation within the cell, and enhances detection accuracy. This provides a highly versatile method for improving sensor performance in the construction of SERS sensors. Summary of the Invention

[0008] In view of this, the present invention aims to explore the dual-function role of Raman dyes in the cell silencing region and construct an i-motif-based SERS sensor that can dynamically monitor changes in intracellular pH. This enables dynamic monitoring of pH in the endosome-lysosome process, improves the stability of the probe in complex environments, achieves controllable aggregation in the intracellular environment, and improves detection accuracy. This method is not only sensitive and accurate, but also simple to operate and has better universality.

[0009] A method for improving the performance of SERS sensors by using bifunctional small molecule-assisted modification. This method selects a suitable Raman reporter molecule, which not only plays the role of Raman signal acquisition in the silent region, but also acts as a blocking agent, effectively stabilizing the stability of the sensor and playing an important dual function in the system. The sensor constructed in this way folds the DNA chain into an i-motif structure in an acidic environment, bringing the distance between gold spheres closer and forming secondary hot spots, which significantly enhances the Raman signal in the silent region of the Raman molecule, enabling dynamic tracking of intracellular pH.

[0010] The sensor comprises an Au-ssDNA complex obtained by co-incubating ss-DNA with gold nanoparticles (AuNPs).

[0011] Furthermore, the particle size of the AuNPs is 10-100nm; preferably, the particle size of the AuNPs is one of 30nm-40nm, 40nm-50nm, 50nm-60nm, 60nm-70nm, 70nm-80nm, and 80nm-90nm.

[0012] Furthermore, the Raman dye for the silent region is a small molecule compound that has both chemical groups that elute in the Raman silent region of the cell and functional groups such as thiol groups that can be stably coupled with gold spheres.

[0013] This invention also discloses a method for preparing a bifunctional small molecule-assisted modified sensor, comprising at least the following steps:

[0014] Gold nanoparticles and thiol-modified i-motifs were coupled via Au-S bonds using a salt aging method to form an Au-i-motif complex.

[0015] Raman dyes were co-incubated with the above-mentioned Au-i-motif complex and attached to the surface of gold spheres via Au-S bonds to prepare SERS probes.

[0016] The present invention also discloses the following process for the above-mentioned sensor to detect pH:

[0017] (1) Disperse probes of equal concentration sequentially in pH buffer solution;

[0018] (2) Add MgCl2 to the buffer solution;

[0019] (3) The absorbance value was measured by ultraviolet spectrophotometer, and a standard curve was established for pH value.

[0020] In step (1), the pH range of the pH buffer is 4.0-7.4.

[0021] In step (2), the concentration of MgCl2 added is 5-30 mM.

[0022] In step (3), the wavelength measured by the ultraviolet spectrophotometer is 798 nm.

[0023] This invention utilizes bifunctional small molecule-assisted modification of gold spheres to construct a pH-responsive SERS sensor. Since the Raman dye in the cell-silenced region also possesses thiol groups, it not only provides Raman signals in the cell-silenced region, improving detection sensitivity, but also acts as a small molecule blocking agent, enhancing sensor stability and preventing non-specific, uncontrolled aggregation in complex cellular environments, thus improving detection accuracy. The i-motif-based SERS sensor prepared using this method exhibits excellent pH responsiveness in the extracellular environment, with the color change resulting from aggregation showing a good linear relationship with pH. Moreover, this response behavior remains unaffected in complex environments, demonstrating good stability. In practical applications, the designed SERS sensor probe can be used for dynamic pH responses in the intracellular environment. The novel i-motif-based SERS sensor, modified with bifunctional small molecules, has the following advantages: 1) high detection sensitivity due to peak elution in the cell Raman-silenced region; 2) good stability and improved detection accuracy; 3) simple operation; 4) good biocompatibility; and 5) rapid response. Therefore, the novel i-motif-based SERS sensor designed with the assistance of bifunctional small molecules has great application potential. Attached Figure Description

[0024] Figure 1 The images show the color, UV, particle size, and potential of the solution before and after i-motif is connected to gold nanospheres.

[0025] Figure 2 The color images of the dye-loaded Au-i-motif probes after 1 h, 2 h, 8 h, and 24 h in PBS, complete culture medium, and FBS are shown.

[0026] Figure 3 The image shows circular dichroisms of i-motif single strands under different pH conditions.

[0027] Figure 4 The images show the color of the Au-i-motif probe loaded with dye under different pH conditions (from left to right, the MgCl2 concentrations in each image are 5, 10, 15, 20, 25, and 30 mM).

[0028] Figure 5 The images show the UV spectra of the probe under pH conditions of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, and 7.4.

[0029] Figure 6 The images are TEM images of the probe at pH 5.0 and different MgCl2 concentrations.

[0030] Figure 7 The images are TEM images of the probe at pH 7.4 and different MgCl2 concentrations.

[0031] Figure 8 The probe reversibly aggregates at different pH values. A) Solution color change graph; B) Ultraviolet graph.

[0032] Figure 9 The images show the Raman spectra of the probe at pH 5.0 and pH 7.4.

[0033] Figure 10 The figures show the cell survival after the probe was incubated with NIH-3T3 and A549 cells for 48 hours.

[0034] Figure 11 The images show the Raman spectra of the probe before and after incubation with A549 and NIH-3T3 cells, respectively.

[0035] Figure 12 Raman images of A549 cells after incubation with the probe for 1-8 hours. Detailed Implementation

[0036] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods, unless otherwise specified, are conventional methods. The present invention will be described in detail below with reference to the embodiments.

[0037] I. Experimental Section

[0038] The names, sequences, and lengths of the DNA in the following examples are shown in Table 1.

[0039] Table 1. Name, sequence, and length of DNA

[0040]

[0041] 1. Preparation of the detection material AuNPs

[0042] Before the experiment, prepare aqua regia for cleaning glass laboratory supplies, using a volume ratio of concentrated hydrochloric acid to concentrated nitric acid of 3:1. It is generally best to use the solution half an hour after preparation.

[0043] 1.1 Synthesis of 60nm Gold Spheres:

[0044] Sodium citrate (4.5 mL, 0.5%) was added to 295 mL of HAuCl4·3H2O aqueous solution (740 μL, 100 mM) in an oil bath with vigorous stirring at 140 °C and stirred for 17 minutes until the solution turned dark red. After natural cooling, the solution was collected and stored at 4 °C.

[0045] Assembly of 1.2-nano gold spheres with single-stranded DNA (ss-DNA):

[0046] The thiol-modified i-motif sequence (5 μL, 100 μM) was reduced using TCEP·HCl (5 μL, 10 mM) at room temperature. The resulting mixture was then added to gold nanoparticles at a molar ratio of 1:100 and reacted overnight for initial ligation. 2 M NaCl was gradually added to the reactants until a final concentration of 0.3 M was achieved, followed by shaking overnight at room temperature. The product was centrifuged at 4 °C (8000 rpm, 5 min) and washed three times with ultrapure water. Finally, the obtained Au-i-motif was redispersed in 1 mL of 0.1% SDS for later use.

[0047] 1.3 Loading of Raman dyes:

[0048] 4-Mercaptobenzonitrile was dissolved in DMSO to prepare a 1 mM solution. 10 μL of the solution was added to the Au-i-motif system and the mixture was shaken at room temperature for 2 h. After that, the mixture was centrifuged (8000 rpm, 5 min) and washed three times with ultrapure water to remove excess Raman dye.

[0049] 2. Probe stability experiment

[0050] The probe was dispersed in PBS, FBS and culture medium respectively, and shaken in a 37°C incubator for 1 h, 2 h, 8 h and 24 h. The state of the solution was observed. If the solution was uniform and clear, it indicated that the probe had good stability.

[0051] 3. pH responsiveness experiment of the probe

[0052] 3.1 Ultraviolet spectrophotometry: The aggregation behavior of the probe was observed by changing two factors: pH and MgCl2 concentration. The probes of equal concentration were dispersed in buffer solutions of pH 4.0-7.4. The final concentration of MgCl2 added to the buffer solution was adjusted to 5-30 mM. The response state of the probe was then observed by ultraviolet spectrophotometer and transmission electron microscope.

[0053] 3.2 Circular dichroism spectroscopy: i-motif single strands were dispersed in PBS (10 mM, pH = 7.4, 15 mM Mg). 2+ In the process, the pH was adjusted to 7.0, 6.5, 6.0, 5.5, and 5.0 with 1M HCl and measured sequentially. The scanning speed was 100 nm / min, the bandwidth was 1 nm, the response time was 1 s, and the scanning wavelength range was 230-340 nm.

[0054] 4. Establishment of the standard curve

[0055] The probes of equal concentration were sequentially dispersed in buffer solutions with pH values ​​of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, and 7.4. The final concentration of MgCl2 added to the buffer solutions was adjusted to 25 mM. The UV absorbance at 798 nm was then measured using a UV spectrophotometer under different pH conditions, and the pH values ​​were plotted.

[0056] 5. Application of practical systems

[0057] 5.1 Cytotoxicity Assay: The cytotoxicity of the Raman probe was detected using the MTT assay. Logarithmic-phase cells were added to 96-well plates (100 μL, 5000 / well) and cultured overnight until cell adhesion. The old culture medium was discarded, and fresh culture medium containing different concentrations of the probe was added sequentially. Cells incubated only with the culture medium served as controls. After 48 h of incubation, 20 μL of MTT (5 mg / mL) solution was added to each well and incubated for 4 h. The culture medium was discarded, and 200 μL of DMSO was added. After shaking for 10 min to fully dissolve the crystal violet, the absorbance was measured at 570 nm using a microplate reader, and the data were processed.

[0058] 5.2 SERS Sample Detection and Cell Imaging Experiments:

[0059] 5.2.1 Sample detection: The liquid sample to be tested was drawn into a capillary tube and measured using a 633nm excitation light source, 10mw laser power, 50× objective lens and 1s acquisition time.

[0060] 5.2.2 Cell Imaging: The cell suspension in the culture dish was injected at a rate of 1 × 10⁶ cells per well. 4 Cells were seeded at a density of 1000 mcg on glass slides and incubated overnight at 37°C and 5% CO2. The Raman probes prepared in Example 1 were added to the cell samples and incubated for different times. The culture medium was then discarded, and the cells were washed three times with PBS to remove free probes and any probes that might be non-specifically attached to the cell surface. The adhered cells were then fixed with 4% paraformaldehyde for 30 min. Finally, the samples were analyzed on a Renishaw Raman spectrometer using a 633 nm excitation source, 10 mW laser power, 50× objective lens, 1 s acquisition time, and 1 μm step size between 1300-2500 cm. -1 Cell imaging is performed within the wavelength range.

[0061] II. Experimental Results and Discussion

[0062] 1. Characterization of the probe

[0063] Gold nanoparticles were prepared and linked to ss-DNA via Au-S bonds using a salt aging method. During this salt aging process, thiol groups gradually replaced the DNA bases adsorbed on the gold nanoparticles, causing the DNA to stand upright on the gold nanoparticle surface. Figure 1As shown in Figure A, no significant color change occurred in the solution before and after the connection. The maximum UV absorption wavelength of the bare gold particle solution, measured using a UV spectrophotometer, was 536 nm. Figure 1 As shown in Figure B, the maximum wavelength of ultraviolet absorption of the modified gold nanoparticle solution undergoes a red shift, reaching 540 nm. Figure 1 The particle size increases after DNA is linked in C. The gold nanoparticles prepared by the sodium citrate reduction method have citrate ions on their surface, giving them a negative charge. The DNA phosphate backbone also carries a negative charge, so the potential becomes more negative after they are linked. Figure 1 The results in D are consistent with this trend, and all of the above results indicate that gold nanoparticles have been successfully linked to DNA.

[0064] Conventional Raman dyes exhibit multiple spectral bands in the fingerprint region, which often overlap with and are difficult to separate from spectral bands derived from dominant endogenous biomolecules. This invention selects small molecules with thiol groups that elute in the cell Raman silencing region as Raman reporter molecules and act as blocking agents. Taking 4-mercaptobenzonitrile as an example, as a Raman dye, the thiol end is tightly connected to the gold bead surface via an Au-S bond, and its nitrile end is located in the cell Raman silencing region (1800-2800 cm⁻¹). -1 A strong and sharp single peak appears at this wavelength, and endogenous biomolecules in the cell do not produce any Raman signal in this wavelength range, thus eliminating background interference.

[0065] 2. Probe biostability

[0066] To verify the contribution of the bifunctional small molecule to the stability of the sensor, the probe was dispersed in PBS (10 mM, pH = 7.4), complete culture medium, and FBS and shaken at 37°C for 1 h, 2 h, 8 h, and 24 h. Figure 2 As shown, the solution color and state did not change significantly with prolonged incubation time, indicating that the prepared nanoprobes are stable in the biological environment and show good application potential.

[0067] 3. Probe pH response characteristics

[0068] Circular dichroism (CD) spectroscopy is an optical rotation spectrum used to infer the configuration and conformation of asymmetric molecules. Optically active substances exhibit circular dichroism because the absorption coefficients (ε) of left-handed and right-handed circularly polarized light (which makes up plane-polarized light) are not equal (εL ≠ εR). If we plot the wavelengths λ of plane-polarized light of different wavelengths on the x-axis and the difference in absorption coefficients Aε = εL - εR on the y-axis, the resulting spectrum is a circular dichroism spectrum, abbreviated as CD. Since Δε can be positive or negative, circular dichroism spectroscopy can also produce positive circular dichroism with peaks and negative circular dichroism with valleys. Figure 3In the circular dichroism spectroscopy (CDS) at pH 7.4, positive and negative peaks were observed at 277 nm and 252 nm, respectively, indicating a normal single-stranded DNA structure. Between pH 7.0 and 6.0, the peak positions did not change significantly, indicating that the DNA remained in an unfolded state. However, when the pH dropped to 5.5, a significant change in the CDS spectrum was observed, with positive and negative peaks appearing at 287 nm and 253 nm, signifying the formation of i-motif secondary structures. Lowering the pH further promoted the formation of secondary structures, as reflected in the increased signal intensity of both positive and negative peaks in the CDS at pH 5.0.

[0069] like Figure 4-5 As shown, different response behaviors of the probe were observed by adjusting two factors: pH and MgCl2 concentration. In the pH range of 4.0-5.5, as the MgCl2 concentration increased, the probe aggregated to varying degrees, which was reflected in the solution color changing from dark red to purple and gray. Gray indicates that the probe aggregated violently, and the system was very unstable at this time. The solution then became almost transparent, and the probe particles that had settled to the bottom were visible to the naked eye. Figure 5 The mid-to-ultraviolet (UV) spectra also confirmed this response process. The SPR peak was highly correlated with the gap distance in the aggregates, and the SPR peak gradually red-shifted as the gap distance decreased. The corresponding peak broadening or red-shifting can be observed in the figure, and the lower the pH of the system, the lower the MgCl2 concentration required for aggregation to begin. In the pH range of 6.0-6.5, even with a MgCl2 concentration of 30 mM, no significant change was observed in the solution color; only peak broadening was seen in the UV spectrum, indicating that the probe aggregation response was very weak within this pH range. Under neutral conditions at pH 7.0 and pH 7.4, no probe aggregation trend was observed in either the solution color or the UV spectrum. These results demonstrate that probe aggregation is driven by i-motif, with acidic pH conditions being the decisive factor. 2+ The process of providing assistance.

[0070] Next, we selected two of the most representative conditions, pH 5.0 and pH 7.4, for more direct observation using transmission electron microscopy, such as... Figure 6-7As shown, under pH 5.0 conditions, when the MgCl2 concentration is in the range of 5-20 mM, although no aggregation was observed through solution color and UV spectrum, the formation of 2-6 different numbers of gold nanoparticles was observed through TEM. When the MgCl2 concentration reaches 25 mM and above, larger aggregates can be observed, which is consistent with the aforementioned experimental results. However, under neutral conditions at pH 7.4, the probe maintains good dispersibility, proving that the sensor has excellent pH response, and the pH inflection point of its i-motif structure change falls precisely within the pH range of lysosomes.

[0071] exist Figure 6 The aggregation behavior assisted by MgCl2 under acidic conditions (pH 5.0) was clearly observed, accompanied by a change in solution color from dark red to purple and then gray. It is important to emphasize that this aggregation is completely different from the irreversible aggregation of gold nanospheres caused by decreased stability that is commonly observed; we conducted reversibility experiments to investigate this. Figure 8 As shown, when the pH of the solution was first adjusted from 7.4 to 5.0 using 1M HCl, the solution color changed from dark red to purple. The UV spectrum also showed a broadening and redshift of the peak at 540 nm, which was the initial peak of maximum absorption. Then, when the pH was adjusted back to 7.4 using 1M NaOH, the solution color changed back to a uniform and transparent dark red. The UV spectrum also showed a return to the peak at 540 nm, indicating that the aggregates were easily broken down and returned to a dispersed state. Note that the solution should not be left for too long when returning from an aggregated to a dispersed state. When the solution turns gray, indicating significant aggregation, the entire system becomes very unstable and cannot be redispersed even after pH adjustment. This reversible aggregation-dispersion behavior suggests that the probe aggregation is actually caused by the formation of pH-induced i-motif structures, rather than by other unstable factors in the system.

[0072] 4. Aggregation-induced Raman hotspot generation

[0073] like Figure 9 As shown, we measured the Raman spectra of the probe under different pH and MgCl2 concentrations. The signal peak of the reported molecule 4-mercaptobenzonitrile (MBN) was assigned to 579 cm⁻¹. -1 Au-CN oscillation, 1074cm -1 CC vibration on the benzene ring, 1118 cm⁻¹ -1 Symmetric tensile vibration of CN, 1580cm -1 Symmetric tensile vibration of benzene ring, 2220 cm-1 C≡N tensile vibration. It can be seen that when the probe is focused, the intensity of the corresponding signal peak is greatly enhanced.

[0074] 5. Cell Raman Imaging

[0075] Before using the probe for dynamic monitoring of lysosomes, its cytotoxicity must first be investigated. For example... Figure 10 As shown, 0.25 μM, 0.5 μM, 0.75 μM, and 1 μM probes were co-incubated with NIH-3T3 and A549 cells for 48 h. When the probe concentration reached 1 μM, the cell viability still reached over 80%, demonstrating that the probe has good biocompatibility. Error bars represent the standard deviation of six independent measurements.

[0076] When probes and cells are co-incubated, the probes enter the cells via endocytosis. As the lysosomes mature, the pH within the subcellular organelles gradually decreases. Under these acidic conditions, the probes aggregate in response, forming Raman hotspots, and the Raman signal of the signaling molecules is significantly enhanced. Figure 11 As shown, by comparing the Raman signal peaks of A549 and NIH-3T3 cells, a new single peak (2220 cm⁻¹) was observed in the Raman silencing region of cells after probe incubation. -1 This peak clearly belongs to the C≡N region. Obviously, in the fingerprint region (e.g., a typical 1580 cm⁻¹), this peak is more pronounced. -1 The probe peaks at the 2220 cm⁻¹ overlap with signals generated by endogenous cytochromes and proteins, especially when they are close to the surface of the gold probe spheres. These complex signals are difficult to resolve, thus producing unavoidable background noise, although at 2220 cm⁻¹... -1 The C≡N peaks can be completely separated from the background signal because endogenous substances do not show any signal in this region. Therefore, we believe that SERS imaging in the cell Raman-silenced region can provide a higher signal-to-noise ratio than in the fingerprint region. Next, a 1900 cm⁻¹ image was selected. -1 Centered on wavelength, 1300-2500cm -1 The wavelength range was measured using a 2220 cm⁻¹ Raman confocal microscope. -1 Channels are used for cell imaging.

[0077] like Figure 12As shown, the probe was first incubated with A549 cells for 1-8 hours, and its dynamic changes were monitored. Throughout the endosome-lysosome pathway, the decrease in pH mediated the degradation of proteins and lipids. Although the pH of the extracellular culture medium and early endosome vesicles was approximately 7.4-7.0, the pH decreased to 6 during endosome maturation, and even to 4.5-5 in lysosomes to support the activity of digestive enzymes. As endosomes matured into lysosomes and the pH decreased, the probe began to respond under acidic conditions, forming aggregates and thus Raman hotspots. A Raman signal was generated after 3 hours of incubation, reaching its strongest point at 4-6 hours, and then showing a decreasing trend after 7-8 hours. This is because during 1-2 hours, the endosomes were not yet mature (pH 6.0-6.5), and the probe did not respond at this time, thus remaining dispersed after vesicle endocytosis. After 3 hours of incubation, the probe began to respond, aggregate, and produce a signal enhancement. At 4-6 hours, the probe reaches the late lysosomal stage. At this point, the lower pH is sufficient to trigger the formation of the i-motif secondary structure. The gold nanospheres approach each other, enhancing surface plasmon resonance and generating a stronger signal. At 7-8 hours, the probe is further transferred to the pH-neutral Golgi apparatus and multivesicles. Based on previous reversibility experiments, we speculate that the aggregated probe redispersed at this time, thus the hotspots disappeared and the signal weakened.

[0078] Three Summaries

[0079] This invention utilizes the auxiliary modification of bifunctional small molecules to prepare an i-motif-based SERS sensor, which improves the sensor's detection sensitivity and stability, thereby achieving better detection accuracy. It realizes the sensor's effective response to pH values, enabling not only pH detection in solution systems but also dynamic monitoring of intracellular pH, providing a new detection method for targeted disease treatment.

[0080] We first assembled ssDNA onto the surface of gold spheres using a salt-aging method, and then used a suitable Raman reporter molecule in the cell silencing region as a blocking agent to easily and conveniently prepare an i-motif-based SERS sensor. Next, the sensor's performance was systematically characterized using circular dichroism spectroscopy, ultraviolet spectrophotometry, colorimetric observation, transmission electron microscopy, and Raman spectroscopy. The results show that the designed sensor exhibits good pH responsiveness, and this pH response is reversible; moreover, complex sample systems do not significantly affect this responsiveness. Since the optimal pH for i-motif structural changes in this sensor falls within the lysosomal pH variation region, it has the function of lysosome tracking, providing a new method for studying the intracellular lysosomal maturation process.

[0081] SERS spectroscopy provides two observable results: (1) the spectral characteristics of SERS, which contain information about the nanoscale chemical environment of gold nanoparticles; and (2) the signal intensity of SERS, which allows monitoring of morphological changes in gold nanostructures, such as aggregate formation, the position of nanoparticles relative to cellular ultrastructures, and the presence and morphology of nanoaggregates within cells. Compared to conventional SERS substrates that form hotspots before analysis, our SERS probes only self-assemble into aggregates under acidic conditions, thus creating Raman hotspots for signal amplification. More importantly, we selected Raman dyes with non-overlapping single peaks in the Raman-silent regions of cells, eliminating background interference typically observed with conventional dyes. This design enables high-resolution imaging, providing opportunities for high signal-to-noise ratio biosensing, especially in complex environments. Intracellular Raman imaging results demonstrate that this sensor can still trace the maturation process of lysosomes in complex intracellular biological environments, unaffected by intracellular endogenous substances, and without uncontrolled non-specific aggregation.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. sequence list <110> Nankai University <120> A method for improving the performance of SERS sensors using bifunctional small molecule-assisted modification <140> 2021109576401 <141> 2021-08-20 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty four <212> DNA <213> i-motif <400> 1 aaaaaaaaaa aaaacccctt cccc 24

Claims

1. An i-motif-based SERS sensor capable of dynamically monitoring changes in intracellular pH, the sensor being fabricated comprising the following steps: (1) Gold nanoparticles and thiol-modified i-motifs were coupled through Au-S bonds to form an Au-i-motif complex by salt aging; (2) Raman dye was co-incubated with the above Au-i-motif complex and linked to the surface of gold spheres via Au-S bonds to prepare a SERS probe, wherein, Raman dyes are small molecule compounds that possess both chemical groups that elute in the Raman silencing region of cells and thiol functional groups that can stably couple with gold spheres.

2. The SERS sensor based on i-motif according to claim 1, characterized in that, When the pH is less than 6, the ssDNA of this sensor folds into an i-motif structure, which reduces the spacing between the gold spheres and causes pH-regulated aggregation behavior.

3. A SERS sensor based on i-motif according to claim 1, characterized in that, The pH detection process of this sensor includes the following steps: (1) Disperse probes of equal concentration sequentially in pH buffer solution; (2) Add MgCl2 to the buffer solution; (3) The absorbance value was measured by ultraviolet spectrophotometer, and a standard curve was established for pH value.

4. A SERS sensor based on i-motif according to claim 1, characterized in that, The pH response behavior of this sensor is a reversible process; when the pH rises above 6.0, the sensor can return to its initial state.

5. A SERS sensor based on i-motif according to claim 1, characterized in that, The sensor's pH response range coincides with the pH variation range of the endosome-lysosome maturation process, and it has the potential to trace lysosomes.

6. A SERS sensor based on i-motif according to claim 1, characterized in that, This sensor can effectively respond to pH changes in the complex intracellular environment and can be used to monitor the intracellular endosome-lysosome maturation process.

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