Part II: Instrumentation & Engineering
Chapter 4: Compensated Interferometric Reader Signal Extraction and Analysis
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Chapter 4: Compensated Interferometric Reader Signal
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period marked in pink under the header B. Next the reference solution fills both detection regions and the phase returns to 0 (time C). At time D, now the reference solution fills detection region 2 and the test sample detection region 1, with the result being a phase change of equal magnitude as in B, but in the opposite direction. Then, following a transition period, test sample 2 fills both detection regions, producing a phase shift of 0, shown as time E in Fig. 4.1. There are 5 regions of interest here: A and E indicate when the sample fills both detection regions, and C indicates when the reference fills both detection regions. It is important to note that the flow is continuous,
Fig. 4.1 The fundamental operation of the differential measurement as the droplet train moves through the two detection regions.
A B C D E
A B C D E
Ti m e
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and for the standard droplet size (1 µL) and flow speed (10 µL/min), each highlighted time section (A-E) corresponds to ~3 seconds.
The phase difference at time A, C, and E should be 0. Reasons for a non-zero measurement in regions A, C, and E are discussed in Appendix C. Times B and D report the differential signal, which is the data needed for the measurement. The average phase change over the duration of B is termed the “test/reference phase shift (+p),” while the average phase change over the duration of D is the “reference/test phase shift (-p).”
Figure 4.2 is a cartoon graphic of the data that would result from 3 test/reference solution pairs, resulting in 3 test/reference phase shifts (+p), 3 negative phase shifts reference/test phase shifts (-p), and 6 regions where the phase shift is 0. As demonstrated in Fig. 4.2, there are several methods that can be employed to obtain a value for the “signal.” The first method, as demonstrated in Fig. 4.2A, is to calculate the phase shift as the test/reference solution phase shift’s excursion from the baseline. Performing the Phase shift – baseline calculation results in an n=3, with an average phase shift of +p (denoted by the three vertical arrows in Fig. 4.2A). A similar phase shift – baseline calculation can also be performed by subtracting the negative going reference/test excursion (For example, Fig. 4.1 region D, -p). Because the same test and reference sample are used to produce the alternating droplets, the phase shift from Fig. 4.1 region D should have the same signal magnitude as region B with opposite sign. The second method, as demonstrated in Fig. 4.2B, is to take the test/reference phase shift and subtract it from the reference/test phase shift (corresponding to the difference of D – E in Fig. 4.1). This results in a signal with magnitude 2×p,
Fig. 4.2 A) Signal quantification by taking the difference between the phase shift and baseline results in an average value of +p with n=3, but quantification of the signal using the difference between phase shifts B) results in an average value of 2×p and an n=5.
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and an n=5, as denoted by the five alternating arrows in Fig. 4.2B. For example, if we apply the strategy described in Fig. 4.2 to the data in Fig. 4.1, +p = 0.48, -p = 0.42, and 2×p = 0.90 radians.
A complete determination in the standard droplet train typically consists of 5 replicate pairs of test/reference droplets (10 droplets total). An example of a complete determination is displayed in Fig. 4.3 for a sample pair consisting of 5 millimolar glycerol in water as the test sample and water with no glycerol as the reference. The data in Fig. 4.3 has the same step-wise pattern correlating to regions A-E as in Fig. 4.1, and the regions are color coded the same way. The data in Fig 4.3 can be tabulated in the +p method, the -p method, and the 2×p method, and these results are presented in Fig. 4.4. As demonstrated in Fig. 4.4, the +p and -p phase shift have approximately the same magnitude (0.214 vs. -0.201) with opposite sign, and similar standard deviation (0.017 vs. 0.021). The 2×p signal, however, is nearly double the +p and -p signal, while the noise (standard deviation) only increases by a factor of ~1.5. The result is a modest, but not insignificant, increase to signal-to-noise (14.3 vs. 12.1 and -9.8). Therefore the 2×p method is superior to the +p or -p method, and unless otherwise stated all further CIR signal determinations use the 2×p method.
Fig. 4.3 Data resulting from 5 test/reference solution pairs consisting of 5 millimolar glycerol in water. The colors correspond to the colors in Fig. 4.1.
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It should be noted here that as demonstrated by the data in Figure 3, a 5 test/reference solution pair results in 19 regions, of which 10 will be “baseline.” The first “baseline” is typically longer than the others, due to the first droplet in the standard droplet train configuration having a larger volume that the other droplets (described in more detail below). Following the initial long baseline, there will be 5 test/reference regions (+p, colored orange) and 4 reference/test regions (- p, colored orange).
The droplet train
The typical FSA-CIR assay consists of preparing a batch of samples that can be used to produce a droplet train. The current instrument incorporates a commercial Dropix droplet generator that employs a sample tray with 14 usable wells that are immersed in an oil bath.
Droplets are made by the capillary moving between the oil and sample reservoir drawing up solutions that are separated by an oil droplet, while the syringe pump provides constant draw. With 14 wells available per tray, the standard assay consists of two rinse solutions and 6 test- solution/reference-solution pairs separated by oil, as presented graphically in Fig. 4.5. As shown in Fig.4.5A, the initial rinses are doubled.
An expanded view of a set of test/reference solution pairs is displayed in Fig 4.5, expressed by showing the time each droplet takes to traverse the detection region. The standard droplet train
Fig. 4.4 The phase shift data in Fig. 4.3, analyzed according to the schema presented in Fig.
4.2. Quantifying the signal as 2×p provides an improvement in S/N over the +p and -p method.
+p -p 2×p
Average 0.214 -0.201 0.408
Standard
Deviation 0.017 0.021 0.028
S/N 12.1 -9.8 14.3
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begins with 2 initial rinses of 2×4 µL droplets of a rinse solution, typically water, followed by a second pair of 2 droplets of 4 µL of a 2nd rinse solution, typically buffer. The volumes and material used for the rinses can be larger in volume and consist of stronger solvents as needed by the assay.
For example, a simple glycerol calibration curve in phosphate buffered saline (PBS) would use PBS as both rinse 1 and 2, but an experiment measuring antibody binding to vesicles may use chloroform as solution 1 and a 25% methanol solution as rinse 2. The combination of rinses can vary according to the experimental matrix (serum, plasma, urine, etc.) and the molecules present in solution (proteins, peptides, lipids, etc.).
The Dropix operating manual describes programing of the Dropix and guides the user on flow rate vs. droplet size. I have employed a syringe pump flow rate of 10 µL/min to generate the droplet train presented here. At this flow rate, the time each droplet takes to traverse in front of a point on the detector is calculated by dividing the volume by the flow rate. For example, the 1000 µL sample droplet moving at 10 µL/min spends 6 seconds in front of a point on the detector. The lengths of all drops used in the standard droplet train are presented in Fig. 4.5. The residence time of a droplet in the detection region is important to calculate, because the output of the interferometer registers or records the signal from the droplets as radians (fringe shift) vs. time.
Fig. 4.5 Droplet train composition by length of time at a fixed point on the detector. A) A single determination consisting of 2 sets of duplicated 4 µL rinse solutions followed by 5 test/reference solution pairs. B) The standard droplet train consisting of 6 sample pair determinations.
A) Single determination
I. Initial Rinses – 98.4 seconds
II. 5 Sample/Reference pairs – 96 seconds
III. Rinses proceeding subsequent Samples – 48.2
9 sec Ref
24 secondsRinse Solution 1 24 secondsRinse Solution 1
6 sec Sam 6 sec
Ref
24 secondsRinse Solution 2 24 secondsRinse Solution 2
6 sec Sam 6 sec
Ref 6 sec
Sam 6 sec
Ref 6 sec Sam 6 sec
Ref 6 sec Sam 6 sec
Oil
24 secondsRinse Solution 1 24 secondsRinse Solution 2
*100 nl oil drops = 0.6 seconds x 4 = 2.4 seconds
*50 nl oil drops = 0.3 seconds x 9 = 2.7 seconds
*100 nl oil drops = 0.6 seconds x 2 = 1.2 seconds Oil
Sample Reference Rinse 2
Rinse 1
Initial Rinse Rinse 2 Rinse 3 Rinse 4 Rinse 5 Rinse 6
Sample pair #1 Sample pair #2 Sample pair #3 Sample pair #4 Sample pair #5 Sample pair #6
B) Complete droplet train
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The code to program the Dropix to deliver the standard droplet train is included in Appendix C.
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