In vitro concentration dependent detection of creatinine: a surface enhanced Raman scattering and fluorescence study† Debraj Gangopadhyay, Poornima Sharma, Rajib Nandi, Moumita Das, Surajit Ghosh and Ranjan K. Singh * An attempt has been made to detect the concentration of creatinine in a very dilute aqueous solution in vitro on the basis of the Jaffe reaction, the commonly used reaction for clinical determination of creatinine concentration in blood serum or urine. For this various spectroscopic techniques viz. Raman spectroscopy, surface enhanced Raman scattering, UV-visible and fluorescence spectroscopy have been used. As a result of the Jaffe reaction using aqueous solutions of creatinine at different concentrations and picric acid and NaOH as reagents, a reddish orange colored Jaffe complex is produced. The concentration dependent SERS spectra of the Jaffe complex show gradual decrease in the intensity of ring deformation mode of creatinine on decreasing creatinine concentration. The concentration dependent absorption spectra of the Jaffe complex show changes in intensity in one of the absorption peaks characteristic of creatinine. The concentration dependent fluorescence emission spectra of the Jaffe complex show a blue shift in emission maxima on reducing the concentration of creatinine in the solution. The observed results suggest the possibility to detect the concentration of creatinine in a very dilute solution in vitro by SERS and fluorescence techniques and the application of these techniques in vivo might aid in a more specific and accurate determination of creatinine in serum or urine. 1. Introduction The measurement of creatinine (CRN) is useful in the deter- mination of renal function and renal health. 1–4 Its normal value in serum is about 0.5–1.5 mg dl 1 . In kidney patients there is an elevation in serum CRN level which is usually accompanied by an elevation in blood urea nitrogen (BUN) levels as well. Clini- cally the estimation of serum CRN is considered superior to that of BUN as a measure of kidney function. The principle of the measurement of CRN in urine and serum is based on the Jaffe reaction which was rst reported by Max Jaffe in 1886 (ref. 5) and was applied by Otto Folin in 1904. 6 The Jaffe reaction states that under alkaline conditions CRN reacts directly with picric acid (PA) forming a reddish orange colored complex. 7 The color change that occurs is directly proportional to the concentration of CRN. In a standard procedure, 0.75 N NaOH and 1% PA at equal volumes are added to an equal volume of the protein free ltrate (PFF) of serum or dilute urine, whence a reddish orange color is developed at room temperature. The colored complex is analyzed by UV-vis absorption or chromatographic techniques to roughly estimate the amount of CRN. However several other substances including proteins, ketones, glucose and ascorbic acid also react with PA producing similar colored complexes 8–12 and the absorbance maxima of these complexes interfered with the absorbance peak of PA–CRN complex. Thus the observed color change is not specically due to CRN and has contribution from other interfering substances also. This has motivated the development of new methods for CRN measurement, 9–12 although almost all such methods are based on some modied form of the Jaffe reaction. The Jaffe reaction, despite its non- specicity for CRN, is still widely employed as the method for CRN measurement 13–20 due to its speed, adaptability in auto- mated analysis and cost-effectiveness. It is the oldest method- ology which is used in the medical laboratory till date. 21 Some of the modications 22–26 made to the standard Jaffe reaction based method for CRN measurement can be divided into three main categories: (i) Estimating the total colored complex (including CRN and non-CRN complexes). (ii) Estimating only true CRN (CRN in serum is directly absorbed into Lloyd's reagent. This is then eluted with alkaline picrate and a red color is developed). (iii) Employing the auto analyzer (where the total number of constituents contributing to the colored complex are uncertain). The mechanism of the Jaffe reaction has been widely investi- gated and a number of structures have been proposed. 27–34 However, controversy still exists about the structure of the reddish Department of Physics, Banaras Hindu University, Varanasi-221005, India. E-mail: ranjanksingh65@rediffmail.com; Tel: +91 5426701569 † Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra22886k Cite this: RSC Adv. , 2016, 6, 112562 Received 13th September 2016 Accepted 22nd November 2016 DOI: 10.1039/c6ra22886k www.rsc.org/advances 112562 | RSC Adv. , 2016, 6, 112562–112567 This journal is © The Royal Society of Chemistry 2016 RSC Advances PAPER Published on 22 November 2016. Downloaded by Banaras Hindu University on 28/02/2017 11:01:16. View Article Online View Journal | View Issue