Application Note # 108

SPR Navi 200 analytical performance

Introduction
A common analytical practice is to determine levels of detection and quantisation, maximum error for repetition from the smallest quantifiable concentration and maximum error for quantisation. The analytical performance of the SPR Navi 200 was studied for these parameters using compounds that have well known refractive index in water solutions. the analyst is linear below 5 wt%. Differences in temperature and wavelength between reference and experimental conditions are neglected in the analysis as we measure changes in RI.

Results and discussion

Results of all experiments were plotted signal versus mass concentration and fitted with RMS linear fit (Figure1 and 2). The linear fit results are collected in Table1 as well as the theoretical dn/dc of the analysts, the signal to RIU conversion coefficients. The data for all experiments corresponds well for linear fit, as the R values in each case are close to one (perfect linear fit). The standard deviation of a fit is the RMS error for the line fit, and it is further used in error calculations. The SD of repetition injections was used to estimate the maximum error of the experiment (as the repetition experiments are one of the smallest ones in the experiments and have therefore the largest possible relative error of the samples). The experimental analytical error was calculated using RMS averaging, and the results are displayed in Table2. LOD and LOQ in terms of analyte RI were calculated from the SD of the baseline of each experiment and converted to RI units by using the conversion coefficient from the linear fits. The results are displayed also in Table2. The LOD and LOQ values for the experiments are excellent, as it shows that we are able to measure analytically meaningful changes in RI in the range of 10-6 at best. The FA mode has smaller level of detection, but it has slightly smaller linearity and larger relative RMS error than AS mode. The FA mode is clearly better in measuring really small changes in RI because of the sensitivity, and the AS mode for experiments where larger dynamic range is needed. The whole range of AS mode was not tried in this experiment, because the dn/dc of the analysts in water used are nonlinear in larger concentrations (over 5 wt%). In experiments in which there is a need for a reference channel correction, the AS mode is more versatile due to its higher dynamic range. This can be useful in biochemical systems where there is a high degree of surface immobilization in one channel, while the other is used to subtract matrix interactions from the injection.

Experimental method
A series of ethylene glycol (EG) concentrations and ethanol concentrations ranging from 5 wt% downwards for ethanol and 2 wt% downwards for EG were diluted in distilled water. Different measurement modes were used for EG and ethanol experiments. EG experiment was done in angular scan mode and ethanol experiments were done in both angular- and fixed angle scan modes (AS and FA, respectively). The measurements were used to create calibration curves, test for repeatability of injections and to measure levels of detection (LOD) and quantisation (LOQ). The measurements were done in ambient conditions. The data was fitted in Origin 7.0 software with least square minimum linear regression method for calibration curve. The calibration curve was evaluated based on the correlation coefficient (R) and standard deviation (SD). The repeatability was tested by five injections of a sample close to the LOQ - limit in order to estimate the maximum error for repeatability (ERMAX) from the SD of the injections. Maximum error for the experiments was estimated by RMS averaging the SDs of the linear fit and repeatability experiment (ETMAX).

Other error sources (sample dilution, purity) were evaluated to be included in the calibration curve error, or to be several orders of magnitude smaller. LOD was defined as three times, and LOQ as five times, of the SD of baseline during the experiments performed. Reference data for refractive index (RI) concentration dependency was taken from CRC [1], and it is assumed that the dn/dc of

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Figure 1. SPR minimum angle change as a function of concentration of ethanol (x) and ethylene glycol (+).

Figure 2. Fixed angle intensity change as a function of ethanol concentration.

Slope(ds/dc)1 EG - AS EtOH - AS EtOH - FA 0,109748 0,06817 0,01914

R 0,9996 0,9998 0,9972

1

SD(fit) 0,00262 0,0019 1,038E-04

Theor(dn/dc)2 9,49E-04 6,00E-04 6,00E-04

Conv coef 3 115,62 113,62 68,14

Table 1. Linear fit data for the experiments. Slope and SD in unit is deg/wt% for AS and RU/wt% for FA. 2Unit for dn/dc is RI/wt%. 3The calibration coefficient is (ds/dc)/(dn/dc) = (ds/dn) in 1/deg for AS and 1/RU for FA.

E (slope)1 EG - AS EtOH - AS EtOH - FA 0,0239 0,0279 0,00542

ERMAX%1 0,0500 0,0462 0,0681

ERMAX%2 5,54 5,40 6,83

SD(bg, sig)3 7,03E-04 1,35E-03 1,62E-05

LOD 0,0021 0,0040 4,848E-05

LOQ 0,0035 0,0067 0,000081

LOD (RIU)4 1,82E-05 3,55E-05 7,11E-07

LOQ (RIU)4 3,04E-05 5,92E-05 1,19E-06

Table 2. 1Relative errors in arbitrary units. 2RMS error in percentages. 3Background SD, in deg for AS and in RU for FA. 4 LOD and LOQ for change in RI for the experiments.

Conclusions
The BioNavis SPR Navi 200 was shown to have excellent analytical performance. It was shown to be able to be used to detect and quantify analytically meaningful changes in the RI in the range of 10-6 RIU. It also showed excellent analytical repeatability and low maximum error, meaning that the instrumental error will not be a deciding factor in an analytical method.
[1] CRC handbook of chemistry and physics, 85th ed., CRC press.

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