MERCADO DE SUBASTAS DE PRODUCTOS AGRÍCOLAS
4.1 Mercado de subastas de productos agrícolas
The strongest band of urea at 1010 cm-1, corresponding to the CN symmetric stretching vibration band, was used to monitor the concentration of urea in the solution.
Fig. 48 shows a comparison of standard and modulated Raman spectra of urea. It can be seen that the modulation technique has significantly flattened the fluorescent background in the spectra and highlighted the Raman peak.
Fig. 48: Comparison of standard and modulated Raman spectra of urea.
To estimate the detection sensitivity of the device, Raman spectra of urea solutions at different concentrations were acquired. 15 sets of Raman spectra were acquired for each concentration. Fig. 49a show a comparison of the standard and modulated Raman spectra acquired for two different concentrations of urea solutions. It was observed that the noise is notably reduced for the modulated Raman spectra of higher concentration urea solutions compared to that of the standard Raman spectra. The SNR of the system at each concentration was evaluated by taking the ratio of the average of peak intensity value (peak to peak value for modulated Raman spectra) to the standard deviation over the spectral region 950 cm-1 to 1050 cm-1. Fig. 49b shows the variation of SNR with concentration for standard Raman spectroscopy and modulated Raman spectroscopy.
The system’s ability to measure concentration relies mainly on two factors: the NEC and the sensitivity. NEC corresponds to the minimum detection limit of the system, which is the concentration at which the SNR becomes equal to unity [136]. In this context, the sensitivity is defined as the slope of concentration vs. SNR curve. NEC depends on such factors as collection efficiency, power of excitation, acquisition time of a single Raman spectra and noise performance of the detector. For both standard and modulated Raman spectroscopy all these parameters are common. Hence it can be seen from Fig. 49b that NEC is same for both standard and modulated Raman spectroscopy.
However, with all the aforementioned parameters fixed, the sensitivity of the device depends on the SNR of the spectra which could be influenced by background fluorescence. It can be seen that there is a 7 fold increase in the slope of the curve (β) for modulated Raman spectra compared to that of the standard Raman spectra in Fig. 49b. It can also be seen in Fig. 49b that, in the region covering normal histological range of human urine, the modulated spectra has a better SNR. This means that the resolution and robustness of WCRS for concentration estimation has been significantly enhanced by the fluorescent suppression provided by this technique.
Fig. 49: [a] Comparison of standard and modulated Raman spectra of urea for 100mM and 450mM concentrations. [b] Urea concentration vs. SNR graph for standard Raman (blue) and modulated Raman (red) spectroscopic methods.
The SNR of the modulated Raman spectra varies with respect to the total acquisition time. From the obtained data the SNR for the 1010 cm-1 peak of urea was calculated for different acquisition times and a fixed concentration (300mM) as shown in Fig. 50a. The modulation frequency and the acquisition time per signal were kept constant and the number of spectra was varied to change the total acquisition time.
At concentrations which lie within the normal histological levels of urea in human urine, the SNR of modulated Raman spectra is better than that of standard Raman spectra with a 5 times longer acquisition time. By directly comparing the standard Raman spectrum obtained with an integration time of 100s and the modulated Raman spectrum obtained with an integration time of only ~20s, it can be clearly observe that the SNR values are comparable. This result demonstrates that the modulated Raman
spectroscopy not only allows removal of the fluorescence background but that it also clearly improves the SNR, reducing the required acquisition time.
Fig. 50: [a] Variation of SNR in relation to the acquisition time for Raman peak of urea at 300 mM concentration in modulated WLRS. [b] Modulated Raman Spectrum of urea obtained with an integration time of 18s and standard Raman spectrum of the same sample obtained with an integration time of 100s.
7.5
Conclusion
Chapter 6 discussed the technology of WCRS which enabled fiber based in situ Raman spectroscopic detection of analytes within a microfluidic chip. WCRS is a generic architecture, readily compatible with various advanced Raman spectroscopic techniques to achieve enhanced detection sensitivity. One such enhancement technique is to suppress the fluorescence background.
In this chapter, wavelength modulation based fluorescent suppression technique was used to enhance the detection sensitivity of a WCRS based bioanalyte sensor. To validate the sensitivity enhancement the bio-analyte detection experiment using urea which was explained in chapter 5 and 6 was repeated with optimized acquisition parameters for the wavelength modulation based fluorescent suppression technique. Compared to standard Raman spectroscopy, the sensitivity of the device showed a 7 fold enhancement when modulated Raman spectroscopy was implemented. Furthermore the variation of the sensitivity of the modulated Raman method with acquisition time was studied and it was found that the SNR for modulated Raman is equivalent to that of a standard Raman spectrum with 5 times larger acquisition time.
Wavelength modulation based Raman spectroscopy has proved to be useful in enhancing the bioanalyte detection sensitivity. However, the total acquisition time required would be higher when implementing wavelength modulation technique since a set of multiple spectra is required. It is also essential that the SNR of the Raman peaks in each single spectrum acquisition should be greater than 1. Hence, as can be seen from Fig. 49b, wavelength modulation based fluorescence suppression technique will not improve the minimum detection limit. However this technique is capable of enhancing the SNR of a Raman peak when the SNR is greater than 1 which is especially useful when multicomponent detection is required from analyte which has relatively strong fluorescence.
In this study the flexibility and compatibility of WCRS with advanced Raman spectroscopic techniques in WCRS was demonstrated. The combination of WCRS with fluorescent suppression techniques opens up opportunities to develop portable microfluidic devices for a wide variety of analyte sensing applications. It may be possible to implement other advanced techniques like SERS or Resonance Raman spectroscopy in conjunction with WCRS to achieve further enhanced detection for specific applications.
Relevant publications
• Ashok PC, Luca ACD, Mazilu M, Dholakia K (2011) Enhanced bioanalyte detection in waveguide confined raman spectroscopy using modulation techniques. J Biophot. doi:10.1002/jbio.201000107
Contributions
P. C. Ashok setup the optics and performed the experiment. A. C. De Luca processed the acquired Raman spectra. M. Mazilu assisted in data processing.