Numerical INner Filter Effect Corrector - NINFE

by Tomislav Friganović, Davor Šakić and Tin Weitner

University of Zagreb, Faculty of Pharmacy and Biochemistry, Zagreb, Croatia.

Help from Mario Gabričević (discussion), Tino Šeba, Valentina Borko, and Robert Kerep (testing) is greatly appreciated. Please report all suggestion, changes, improvements, and usage to davor.sakic@pharma.unizg.hr.

Finanancial support provided through Croatian Science Foundation grant no. UIP-2017-05-9537 "Glycosylation as a factor in the iron transport mechanism of human serum transferrin" GlyMech at University of Zagreb Faculty of Pharmacy and Biochemistry.

Introduction

This is a quick guide in using Numerical INner Filter Effect Corrector - NINFE. Although different methods are available for countering IFE, this method can be implemented into laboratory workflow with minimal hassle.

User guide

Necessary steps:

1. Determine the geometric parameter for the respective microplate reader and microplate type.
2. Measure fluorescence for known fluorophore concentration series to be used for NINFE calibaration. At least two fluorescence measurements at two different z-axis positions (z-positions) for the same sample in the microplate well are required.
3. Format calibration data table (see details below).
4. Run NINFE calibration routine to obtain optimal z-position combination and linear response parameters.
5. Measure fluorescence for samples with unknown fluorophore concentration at the determined optimal z-position combination.
6. Format concentration readout data table (see details below).
7. Run NINFE readout routine to retrieve unknown fluorophore concentrations. The NINFE calibration routine needs to be performed previously. Readout can be used multiple times while using the same calibration.
8. Click START AGAIN for another cycle.

Data formats (input/output)

Input format (calibration):
c z1 z2 z3 z4 z5
uM 14,000 15,000 16,000 17,000 18,000
1.0 10 12 14 16 18
5.0 50 60 70 80 90
10.0 100 120 140 160 180
15.0 150 180 210 240 270
20.0 200 240 280 320 360

Output format (calibration):
ID pair F1 F2 conc1(1.0) conc2(5.0) conc3(10.0) conc4(15.0) conc5(20.0) slope intercept r2 rmsre steyx LOD %mErr BBm/Pm exp
0 0 14,000 15,000 221.861 1,109.306 2,218.611 3,327.917 4,437.222 221.861 -0 1 0 NaN NaN -0 BBm 17
1 0 14,000 15,000 27.724 138.622 277.243 415.865 554.486 27.724 -0 1 0 0 0 0 Pm 5.593
2 1 14,000 16,000 0.012 0.06 0.12 0.179 0.239 0.012 -0 1 0 NaN NaN 0 BBm -20
3 1 14,000 16,000 21.656 108.281 216.562 324.844 433.125 21.656 0 1 0 0 0 0 Pm 2.297
*BBM = Black-box model, Pm = Parametric model

Input format (readout):

designation z1 z2 z3 z4 z5
num 14,000 15,000 16,000 17,000 18,000
A1 10 12 14 16 18
B2 50 60 70 80 90
C3 100 120 140 160 180
D4 150 180 210 240 270
E5 200 240 280 320 360
*Designation = user-defined or sample-specific label, e.g. well position etc.

Output format (readout):

-Parametric model (Pm)
F(15000) F(16000) Fcorrected Pm conc Pm (uM)
1 10 18 14.638 1
2 50 90 73.19 5
3 100 180 146.38 10
4 150 270 219.569 15
5 200 360 292.759 20

-Black-box model (BBm)
F(15000) F(17000) Fcorrected BBm conc BBm (uM)
1 12 14 48.051 1
2 60 70 240.255 5
3 120 140 480.51 10
4 180 210 720.765 15
5 240 280 961.02 20