Quantitative SERS Detection of Uric Acid via Formation of Precise Plasmonic Nanojunctions within Aggregates of Gold Nanoparticles and Cucurbit[n]uril

Podsumowanie

A host-guest complex of cucurbit[7]uril and uric acid was formed in an aqueous solution before adding a small amount into Au NP solution for quantitative surface-enhanced Raman spectroscopy (SERS) sensing using a modular spectrometer.

Streszczenie

This work describes a rapid and highly sensitive method for the quantitative detection of an important biomarker, uric acid (UA), via surface-enhanced Raman spectroscopy (SERS) with a low detection limit of ~0.2 μM for multiple characteristic peaks in the fingerprint region, using a modular spectrometer. This biosensing scheme is mediated by the host-guest complexation between a macrocycle, cucurbit[7]uril (CB7), and UA, and the subsequent formation of precise plasmonic nanojunctions within the self-assembled Au NP: CB7 nanoaggregates. A facile Au NP synthesis of desirable sizes for SERS substrates has also been performed based on the classical citrate-reduction approach with an option to be facilitated using a lab-built automated synthesizer. This protocol can be readily extended to multiplexed detection of biomarkers in body fluids for clinical applications.

Wprowadzenie

Uric acid, which is the end product of metabolism of purine nucleotides, is an important biomarker in blood serum and urine for the diagnosis of diseases such as gout, preeclampsia, renal diseases, hypertension, cardiovascular diseases and diabetes^1,2,3,4,5. Current methods for uric acid detection include colorimetric enzymatic assays, high performance liquid chromatography and capillary electrophoresis, which are time-consuming, expensive and require sophisticated sample preparation^6,7,8,9.

Surface-enhanced Raman spectroscopy is a promising technique for routine point-of-care diagnosis as it allows selective detection of biomolecules via their vibration fingerprints and offers numerous advantages such as high-sensitivity, rapid response, ease of use and no or minimal sample preparation. SERS substrates based on noble metal nanoparticles (e.g., Au NPs) can enhance the Raman signals of the analyte molecules by 4 to 10 orders of magnitude¹⁰ via strong electromagnetic enhancement caused by surface plasmon resonance¹¹. Au NPs of tailored sizes can be easily synthesized as opposed to the time-consuming fabrication of complex metal nanocomposites¹², and thus are widely used in biomedical applications owing to their superior properties^13,14,15,16. Attachment of macrocyclic molecules, cucurbit[n]urils (CBn, where n = 5-8, 10), onto the surface of Au NPs can further enhance the SERS signals of the analyte molecules as the highly symmetric and rigid CB molecules can control the precise spacing between the Au NPs and localize the analyte molecules at the center or in close proximity to the plasmonic hotspots via formation of host-guest complexes (Figure 1)^17,18,19,20. Previous examples of SERS studies using Au NP: CBn nanoaggregates include nitroexplosives, polycyclic aromatics, diaminostilbene, neurotransmitters and creatinine^{21,22,23,24,25}, with the SERS measurements either being performed in a cuvette or by loading a small droplet onto a custom-made sample holder. This detection scheme is particularly useful to rapidly quantify biomarkers in a complex matrix with a high reproducibility.

Herein, a facile method to form host-guest complexes of CB7 and an important biomarker UA, and to quantify UA with a detection limit of 0.2 μM via CB7-mediated aggregations of Au NPs in aqueous media was demonstrated using a modular spectrometer, which is promising for diagnostic and clinical applications.

Protokół

1. Synthesis of Au NPs

1. Synthesis of Au seeds via the conventional Turkevich method²⁶
   1. Prepare 10 mL of 25 mM HAuCl₄ solution by dissolving 98.5 mg of HAuCl₄· 3H₂O precursor with 10 mL of deionized water in a glass vial.
      NOTE: Transfer a small amount of HAuCl₄ precursor into a weighing boat and use a plastic spatula instead of metallic spatula to weigh out the crystals because HAuCl₄ precursor will corrode metal labware. The weighing step should be performed as swiftly as possible, since HAuCl₄ is hygroscopic and will therefore increase its weight over time by absorbing water from the atmosphere. HAuCl₄ is highly corrosive and can cause severe skin burns and eye damage. Take extra care when handling it.
   2. Prepare 0.5 mL of 500 mM sodium citrate solution by dissolving 64.5 mg of sodium citrate powder with 0.5 mL of deionized water in a glass vial.
   3. Dilute 1 mL of the 25 mM HAuCl₄ solution with 99 mL water in a 250 mL blue-capped bottle to give 100 mL of 0.25 mM HAuCl₄ solution.
   4. Add 99.5 mL of the 0.25 mM HAuCl₄ solution into a 250 mL three-necked round-bottomed flask equipped with a condenser. Heat the solution to 90 °C under vigorous stirring and maintain the temperature for 15 min.
   5. Inject 0.5 mL of the 500 mM sodium citrate solution into the reaction mixture and maintain the temperature and stirring until the color of the solution turns ruby-red.
      NOTE: The reaction takes about 30 min.
2. Seeded growth of Au NPs via the kinetically-controlled method¹³
   1. Cool the as-synthesized Au seed solution to 70 °C.
   2. Prepare 10 mL of 60 mM sodium citrate solution by dissolving 154.8 mg of sodium citrate powder with 10 mL of deionized water in a glass vial.
   3. Inject 0.67 mL of the 25 mM HAuCl₄ solution and 0.67 mL of the 60 mM sodium citrate solution to the Au seeds with a time interval of 2 min.
   4. Repeat step 1.2.3 to gradually increase the size of Au NPs to 40 nm.
      NOTE: It takes about 10 growing steps to reach 40 nm. The actual number of steps needed may be dependent on the precise set-up.
3. Seeded growth of Au NPs using automated synthesizer (Figure 2)
   1. Transfer 25 mL of the Au seed solution prepared in section 1 to a 50 mL conical centrifuge tube and cool to 70 °C in a thermomixer.
      NOTE: Monitor the temperature inside the thermomixer using a thermocouple thermometer placed in a 50 mL centrifuge tube containing 25 mL of water.
   2. Fill a 3 mL Luer lock disposable syringe with 2.5 mL of 25 mM HAuCl₄ solution. Fill another 3 mL Luer lock disposable syringe with 2.5 mL of 60 mM sodium citrate solution.
   3. Place the syringes in the syringe pumps and use Luer-to-MicroTight adapters to connect the PEEK tubing (150 µm internal diameter) to the syringes. Insert the tubing into the centrifuge tube containing the Au seed solution in the thermomixer.
   4. Set both syringe pumps to dispense 0.1675 mL of solution over 20 min (8.357 μL per min).
   5. Set the thermomixer rotation speed to 700 rpm and press Start on the syringe pump containing the 25 mM HAuCl₄ solution.
   6. After 2 min, press Start on the syringe pump containing the 60 mM sodium citrate solution.
   7. 30 min after starting the HAuCl₄ solution injection, remove an aliquot of the Au NP solution for analysis.
   8. Repeat steps 1.3.5 – 1.3.7 to gradually increase the diameter of the Au NPs up to 40 nm.
      NOTE: This setup can be used to grow Au NPs up to 40 nm in one step by increasing the volume of reactants added in step 1.3.4. This is achieved by increasing the dispensing time whilst maintaining the same rate of injection.

2. Characterization of Au NPs

1. UV-Vis spectroscopy
   1. Add 1 mL of the Au NP solution to a semi-micro quartz cuvette.
   2. Turn on the spectrometer.
   3. Set the wavelength range to 400 - 800 nm.
   4. Acquire the UV-Vis spectrum for each sample.
2. Dynamic light scattering (DLS)
   1. Filter the sample solution into a plastic semi-micro cuvette with a 0.22 μm filter.
   2. Turn on the DLS instrument.
   3. Set the temperature to 25 °C and equilibrate for 60 s.
   4. Measure the hydrodynamic size of each sample.
3. Transmission electron microscopy (TEM)
   1. Drop-cast a 5 μL droplet of the sample solution onto a C-coated 300-mesh Cu grid and dry in air.
      NOTE: Dilution is needed for more concentrated Au NP solution samples to obtain well dispersed Au NPs on a TEM grid.
   2. Acquire multiple TEM images for each sample using a TEM at 200 kV acceleration voltage.
   3. Measure the diameter of 200 Au NPs for each sample using ImageJ to calculate the average size and standard deviation.

3. Formation of CB7-UA complexes

1. Preparation of 0.4 mM CB7 solution
   1. Add 4.65 mg of CB7 to a 15 mL glass vial.
      NOTE: The amount of CB7 is calculated based on the formula weight of CB7 (= 1163 Da) which has been employed by most reports in the literature. Nevertheless, CB7 solid samples typically contain water, HCl, methanol and other salts left from the synthesis and purification steps, contributing to ~10 – 20% dead weight in the sample. The trapped solvents and salts could not be removed by heating in a vacuum oven or other means. Their amounts vary between different batches of samples but can be quantified using elemental analysis. Yet, the presented protocol is not sensitive to the presence of unquantified amount of solvents and salts in the CB7 samples.
   2. Add 10 mL of water to the vial and tighten the cap.
   3. Sonicate the sample at room temperature until the CB7 solid is completely dissolved.
      NOTE: CB7 was synthesized according to literature²⁷ but it is also commercially available.
2. Preparation of 0.4 mM UA solution
   1. Add 2.69 mg of UA to a 50 mL centrifuge tube.
   2. Add 40 mL of water to the tube and tighten the cap.
   3. Use a thermomixer to swirl the sample solution by setting the temperature to 70 °C, speed to 800 rpm and time to 2 h. Allow the solution to cool to room temperature.
      NOTE: UA has a low solubility in water (0.40 mM)⁵. Swirl for longer if the UA powder has not been dissolved completely. Alternatively, ultrasonication can be used to facilitate the dissolution.
3. Sequential dilutions of the 0.4 mM UA solution
   1. Dilute 5 mL of the 0.4 mM UA solution with 5 mL water in a 15 mL glass vial to give 10 mL of 0.2 mM UA solution. Tighten the cap and sonicate for 30 s.
   2. Repeat step 3.3.1 using an appropriate amount of UA and water as described in Table 1.
4. Preparation of the CB7-UA complexes
   1. Add 0.75 mL of the 0.4 mM CB7 solution and 0.75 mL of 0.4 mM UA solution into a 1.5 mL tube. Secure the lid and sonicate for 30 s.
   2. Wait for 30 min to ensure the formation of host-guest complexes.
   3. Repeat step 3.4.1 – 3.4.2 using UA solution of different concentrations.

4. SERS sensing of UA

1. Experimental set-up of the Raman system (Figure 3)
   1. Switch on the 633 nm He-Ne laser (22.5 mW).
   2. Switch on the modular Raman spectrometer.
   3. Switch on the computer and start the software.
   4. Click the Spectroscopy Application Wizards Icon, and then select Raman.
   5. Start a new acquisition. Set the integration time to 30 s, scans to average to 5 and boxcar to 0.
   6. Store background spectrum and enter the laser wavelength (i.e., 633 nm).
      NOTE: Integration time is the time for each scan, scans to average is number of scans averaged to create each spectrum and boxcar is the number of neighboring pixels averaged²⁸.
2. Formation of the SERS substrates
   1. Add 0.9 mL of the 40 nm Au NP solution and 0.1 mL of the pre-formed CB7-UA complex solution into a 1.5 mL tube. Secure the lid and sonicate until the solution changes from ruby-red to purple.
      NOTE: Commercial citrate-stabilized 40 nm Au NP solution samples can also be used. Typically, the optical density of the localized surface plasmon resonance (LSPR) peak is adjusted to 1 via dilution from concentrated stock solution samples. Citrate concentration in the sample is typically kept as 2 mM.
   2. Transfer the sample solution to a semi-micro cuvette. Place the cuvette into the Raman sample holder and close the cover.
   3. Start the measurement.
   4. Set up the auto-saving to record five consecutive SERS spectra.
   5. Stop the measurement and change the sample.
   6. Repeat step 4.2.1 – 4.2.5 using CB7-UA solution of different concentrations.
      NOTE: Aggregation time is found to be dependent on the concentration of UA in the nanoaggregates, ranging from 30 s for 0.1 μM UA to 30 min for 20 μM UA, owing to the difference in the concentration of empty CB7 which has major contribution to mediating the aggregation of Au NPs. For the CB7-UA complex, one portal is blocked by the bulky UA molecule, rendering it unavailable for binding to the Au NP surface and therefore unable to mediate the NP aggregation²¹. The sample is ready for measurement when the color of the solution changes from ruby-red to purple.

5. Data analysis

1. Data processing
   1. Download and install the baseline with asymmetric least squares (ALS) plugin into Origin.
      NOTE: The ALS plugin requires OriginPro.
   2. Insert the raw data into Origin.
   3. Calculate an average value from the five SERS spectra of each sample. Divide the value by the power of the laser (i.e., 22.5 mW) and by the integration time (i.e., 30 s).
   4. Click the ALS icon to open the dialog. Set the asymmetric factor to 0.001, threshold to 0.03 %, smoothing factor to 2 and number of iterations to 20 to correct the baseline of each averaged spectrum.
   5. Plot the SERS spectra of different UA concentrations using stacked lines by y offsets. The output should be intensity (counts s^-1 mW^-1) against Raman shift (cm^-1).

Wyniki

In the presented Au NP synthesis, the UV-Vis spectra show a shift of the LSPR peaks from 521 nm to 529 nm after 10 growing steps (Figure 4A,B) while the DLS data shows a narrow size distribution as the size of Au NPs increase from 25.9 nm to 42.8 nm (Figure 4C,D). The average sizes of G0, G5 and G10 measured from TEM images (Figure 4E) are 20.1 ± 2.1 nm, 32.5 ± 2.3 nm and 40.0 ± 2.2 n...

Dyskusje

The automated synthesis method described in the protocol allows Au NPs of increasing sizes to be reproducibly synthesized. Although there are some elements that still need to be carried out manually, such as the fast addition of sodium citrate during the seed synthesis and checking periodically to ensure that the PEEK tubing is secure, this method allows Au NPs of large sizes (up to 40 nm), which would usually require multiple manual injections of HAuCl₄ and sodium citrate, to be synthesized via continuous add...

Materiały

Name	Company	Catalog Number	Comments
40 nm gold nanoparticles	NanoComposix	AUCN40-100M	NanoXact, 0.05 mg/ mL, bare (citrate)
Centrifuge tube	Corning Falcon	14-432-22	50 mL volume
Cucurbit[7]uril	Lab-made		see ref. 19
Gold(III) chloride trihydrate	Sigma aldrich	520918	≥99.9% trace metals basis
Luer lock disposable syringe	Cole-Parmer	WZ-07945-15	3 mL volume
Luer-to-MicroTight adapter	LuerTight	P-662	360 μm outer diameter Tubing to Luer Syringe
PEEK tubing	IDEX	1572	360 μm outer diameter, 150 μm inner diameter
PEEK tubing cutter	IDEX	WZ-02013-30	Capillary Polymer Chromatography Tubing Cutter For 360 µm to 1/32" OD tubing
Raman spectrometer	Ocean Optics	QE pro
Sodium citrate tribasic dihydrate	Sigma aldrich	S4641	ACS reagent, ≥99.0%
Sonicator
Standard Probe	Digi-Sense	WZ-08516-55	Type-K
Syringe pump	Aladdin	ALADDIN2-220	2 syringes, maximum syringe volume 60 mL
Thermocouple thermometer	Digi-Sense	WZ-20250-91	Single-Input Thermocouple Thermometer with NIST-Traceable Calibration
ThermoMixer	Eppendorf	5382000031	With an Eppendorf SmartBlock for 50 mL tubes
Uric acid	Sigma aldrich	U2625	≥99%, crystalline