Application Note

Performance of Estapor® RedIntense Microspheres
Comparison study with recognized competitor
microspheres in a model HBsAg antigen assay

Summary
Next generation lateral flow tests (LFTs) mustbe highly sensitive to allow early detection ofpathogens, easy to use (rapid and readable) toallow for home utilization and cost effective to allow
their distribution at mass scale. Moreover, the trend
now is the use of microspheres for multiplex LFTs
which use various colored microspheres to indicate
the presence of multiple analytes.
To address these challenges, we are developing
a new portfolio of intense-colored, carboxylated
microspheres featuring the following
unique advantages:• Improved assay sensitivity through greater
signal intensity• Easy reading, even at low
conjugate concentration• Stable conjugates through easy covalent binding
to the carboxyl-modified microspheres• Reproducible results due to high monodispersity
and color stability• Cost effective• Versatile with a range of 2 diameters available at
300 nm and 400 nm• Available currently in intense red and blue withmore colors in development

Introduction
Lateral flow tests (LFTs) (Figure 1) are simple,rapid, and cost-effective technology for diagnostics
applications. LFT-based assays are used in home,
hospitals, physician’s offices, and clinical labs for thedetection of specific antigens and antibodies.

The Life Science business of Merck
operates as MilliporeSigma in theU.S. and Canada.

Figure 1. Lateral flow test strip design

The massive use of LFTs during the COVID-19
pandemic demonstrates their acceptability by alarge range of the worldwide population, thus
creating a demand for the development of next
generation designs.
Next generation LFTs must be highly sensitive toallow early detection of pathogens, easy to use (rapidand readable) to allow for home utilization and costeffective for distribution at mass scale. There isincreased demand for use of colored microspheresfor multiplex LFTs to indicate the presence of multiple
analytes, for example to differentiate virus strains.
To address these challenges, our Estapor® R&D scientists
and engineers are developing a new set of intensecolored microspheres for use as highly sensitive detector
reagents. The first of this new portfolio available areEstapor® K1-030 Red Intense and K1-040 Red Intense(Table 1), highly monodispersed (Figure 2), carboxyl
modified polystyrene microspheres with a diameter of
300 nm or 400 nm.
In this report, we compare the applicative performance of
the Estapor® Red Intense microspheres with recognizedcompetitor microspheres in a model HBsAg antigen assay.

Table 1: Estapor® Red Intense microspheres
K1-030 Red Intense

K1-040 Red Intense

Catalog number

FR180380637

FR180380638

Functional group

COOH

COOH

Diameter (range)

0.270 - 0.330 μm

0.340 - 0.440 μm

COOH content (range)

200 – 500 μeq/g

10 – 100 μeq/g

Solid concentration

5%5%Polymer

Polystyrene

Polystyrene
ng/mL

ng/mL

ng/mL

ng/mL

ng/mL

ng/mL

Figure 2: High monodispersity illustrated by TEM analysis ofEstapor® K1-030 Red Intense (left) and K1-040 Red Intense (right)

Results and discussion
To illustrate the performance of Estapor Red Intense
microspheres, we have selected HBsAg antigen
detection as a model assay. Microspheres wereconjugated to antibodies using our standard twostep EDC/Sulfo-NHS covalent coupling process (seeExperimental Section for detailed procedure).
®

The new reference K1-040 Red Intense was comparedwith key competitor microspheres, widely used as
reference detector reagent in LFTs. For this purpose,two different carboxyl modified microspheres (noted Aand B) with same size of 400 nm and color were tested
using the same assay conditions.
A first set of experiments was run with a conjugate
concentration of 0.005% (Figure 3 – upper row). At thisconjugate concentration, all tests show readable test andcontrol lines, but Estapor® K1-040 Red Intense gave themost intense signal making the result easier to read.

Figure 3. Comparison of Estapor® Red Intense microspheres andkey competitor A and B. (bead diameter: 400 nm). Upper row:
0.005% conjugate - Lower row: 0.002% conjugate(CL = Control line; TL = Test line)

Estapor® Red Intense microspheres are offered in two
different sizes. The same HBsAg detection model wasused to compare the performance of K1-030 Red Intense(300 nm) to the previously studied K1-040 Red Intense(400 nm) (Figure 4). Both references give intense signal
and show the same level of sensitivity. In our model,K1-030 Red Intense gave slightly less intense lines but
the signal appeared faster than the larger K1-040 RedIntense. Both microsphere sizes performed excellently at
low conjugate concentration.

To further assess the gain of sensitivity obtained using
Red Intense microspheres, we decreased the conjugate
concentration down to 0.002% (Figure 3 – lowerrow). At this concentration, only K1-040 Red Intense
still gives a clearly readable signal where competitor
microspheres signal is too weak.
ng/mL

ng/mL

ng/mL

ng/mL

Figure 4. Comparison of Estapor® K1-030 Red Intense (300 nm)
and K1-040 Red Intense (400 nm) at 0.005% or 0.002% conjugate
concentration (CL = Control line; TL = Test line)2Conclusion
K1-030 Red Intense and K1-040 Red Intense are partof the new line of Estapor® Intense microspheresreferences. Their key advantages are high color
intensity, high monodispersity, and carboxyl
surface modification.
These unique features greatly improve the sensitivityof the lateral flow tests compared to key competitorson the market. The high intensity of the signal opens
the possibility to use lower conjugate concentrations,making the tests more cost effective by decreasing theamount of all raw materials involved.
As such, K1-030 Red Intense and K1-040 Red Intense
are versatile solutions to address the challenges for thedevelopment of next generation LTFs.
The new line of Estapor® Color Intense microspheres
includes Blue Intense and will soon be enrichedwith Black Intense, opening great horizons for thedevelopment of highly sensitive, multiplexed LTFs.

Experimental Section
Methods
Lateral flow test strips for detection of HBsAg
antigen were prepared using Estapor® Red Intense
microspheres or competitor particules, Hi-Flow™ Plus
membrane and anti-HBsAg antibodies. Microspheres
were conjugated to antibodies using a two-step EDC/
Sulfo-NHS covalent coupling process.

PBS

Sigma-Aldrich®, D8537

Polyvinylpyrrolidone (PVP)

Sigma-Aldrich®, PVP10

SucroseSigma-Aldrich®, PG80271

Sulfo-NHS

ThermoFisher, 22980

D-(+)-Trehalose dihydrate

Sigma-Aldrich®, T5251

Tween 20

Sigma-Aldrich®, P7949

Tris

Sigma-Aldrich®, T1378®

BuffersTable 3. Buffers and composition
Activation buffer/
Coupling buffer

50 mM MES, pH 6.0 (pH adjusted withNaOH solution)

Blocking buffer

50 mM Tris, pH 8.0, 0.5% (w/v) casein(pH adjusted with 4 M HCl)

Conjugate padbuffer

50 mM Tris, pH 8.0, 0.5% (w/v) casein,
10% Sucrose, 25% Trehalose, 0.5% PVP(pH adjusted with 4 M HCl)

Sample pad buffer

10 mM Tris, 1% Tween® 20, 0.75% BSA pH 8.2(pH adjusted with 4 M HCl)

Conjugation Protocol
Microsphere Washing and Activation
1.
2.
3.
4.
5.

Equipment• Salvis LAB Vacucenter oven
• Centrifuge 5430
• Kinematic Matrix 1600 Reagent Dispensing Module• Kinematic Matrix 2360 Programmable Shear• Kinematic Matrix 1201 Membrane cutter• Stuart Rotator SB3 Rotary wheel• Vortex-Genie 2 Vortex mixer• Sonicator

Materials & Reagents
Table 2. Reagent and components
Adhesive backing cards

Millipore®, HF000MC100

Analyte HBsAg

Prospec, HBS-875

Bovine Serum Albumin

Sigma-Aldrich®, A7030

Capture Antibody

Creative Diagnostics, DMAB3535

CaseinSigma-Aldrich®, C7078

Cellulose fibre padMillipore®, CFSP 223000Conjugate padMillipore®, GFDX203000

Detector Antibody

Creative Diagnostics, DMAB3534

EDC

ThermoFisher, 77149

Estapor® Red IntenseMicrospheres

Sigma-Aldrich®, FR180380638,
FR180380637

Ethanolamine

Sigma-Aldrich®, 411000

Goat Anti-Mouse IgG

Sigma-Aldrich®, M8890

Hi-Flow™ Plus 135 membrane

Millipore®, HF13502XSS

Hydrochloric Acid (HCl)Sigma-Aldrich®, 320331

MES

Sigma-Aldrich®, M3671

6.

7.
8.
9.

Aliquot 5 mg of Estapor® Red Intense microspheres(5% w/v) into a low-protein binding Eppendorf tube.
Add 1 mL of activation/coupling buffer andmix thoroughly.
Spin the microspheres at 12,000 rpm (~14,200 g)
centrifuge cycle for 7 minutes.
Decant the supernatant.
Repeat steps 2 through 4 twice. It is importantthat the microspheres are completely resuspended
between washes and before progressing with ligand
coating. Sonicate using an ultrasonic probe.
After the final wash, resuspend the microspheres in
1 mL of activation/coupling buffer. It is important
to ensure the microspheres are monodispersed.
Prepare activation reagents immediately prior to use.
To prepare 200 mM EDC solution, add 19.2 mgof EDC at room temperature to 500 µL of Milli-Q®
water in a clean microfuge tube.
To prepare 200 mM Sulfo-NHS, add 21.7 mg ofSulfo-NHS to 500 µL of activation/coupling buffer ina clean microfuge tube.

10. To 1 mL of washed microspheres (from Step 6above) quickly add 12 µL of 200 mM EDC and120 µL of 200 mM Sulfo-NHS.
11. Vortex and incubate by mixing on a rotary wheelfor 30 minutes at room temperature.
12. Spin the microspheres at 12,000 rpm (~14,200 g)
centrifuge cycle for 7 minutes.
13. Decant the supernatant.
14. Add 1 mL of activation/coupling buffer to wash themicrospheres and mix thoroughly.
15. Spin the microspheres at 12,000 rpm (~14,200 g)
centrifuge cycle for 7 minutes.
16. Repeat steps 13 to 15 twice.
17. Resuspend the microspheres in 850 µL ofactivation/ coupling buffer (See antibody
coupling section below). It is important that themicrospheres are monodisperse before adding
antibodies/ligand. Sonicate, if necessary.
3Antibody Coupling

Antibody striping

18. Prepare the antibody at a concentration of 2 mg/mLin activation/coupling buffer.
19. Based on a coating concentration of 60 mg
antibody per gram of microspheres, add
150 µL of antibody (at 2 mg/mL) to the 850 µLof microspheres. Mix thoroughly. The total liquidvolume will now be 1 mL.
20. Mix the suspension for 2.5 hours on a rotary wheel
at room temperature.
21. For 1 mL of microspheres, add 15 µL ofethanolamine (in a fume hood). Vortex and mix
on a rotary wheel for 30 minutes to quench
the microspheres.
22. Spin the microspheres at 12,000 rpm (~14,200 g)centrifuge cycle for 7 minutes.
23. Decant the supernatant.
24. Resuspend the microspheres in 1 mL of blocking
buffer. Sonicate and mix for a minimum of 2 hours
on a rotary wheel at room temperature. Note:
The microspheres can also be left mixing at room
temperature overnight.
25. Spin the microspheres at 12,000 rpm (~14,200 g)
centrifuge cycle for 7 minutes.
26. Decant the supernatant.
27. Add 1 mL of blocking buffer and resuspend themicrospheres by repeated pipetting and sonication,if necessary.
28. Spin the microspheres at 12,000 rpm (~14,200 g)
centrifuge cycle for 7 minutes.
29. Repeat steps 26 to 28 once more.
30. After the final wash step, remove the supernatantand add 1 mL of blocking buffer. The microspheres
are now at 0.5% w/v.
31. Resuspend the microspheres using repeatedpipetting and sonication. Optional: Examine themicrospheres under a microscope to ensure thatthey are monodisperse.
32. Store the suspension at 4 °C until ready to use.

36. Dilute the control line reagent goat anti-mouse to1 mg/mL using Milli-Q® water.
37. Prepare the test line reagent at a concentration of
1 mg/mL using Milli-Q®.
38. Antibodies were striped using Kinematic Matrix
1600 Reagent Dispensing Module at the rate of
1 µL/cm with 10 cm/sec line speed.
39. Dry the striped membrane at 37 °C for 2 hours
using Salvis LAB Vacucenter oven.
Test strip preparation and Testing
40. Stripe the antibodies using Kinematic Matrix 2360
Programmable Shear.
41. Analytes were prepared at concentration of 0, 8,20, 60, 240 ng/mL using 1:1 PBS:Serum.
42. Add 150 µL serum to the sample pad on the teststrip and analyse the result 15-30 min.

References1.

Budd, J., Miller, B.S., Weckman, N.E. et al. (2023). Lateral flow
test engineering and lessons learned from COVID-19. Nature
Reviews Bioengineering.

2.

Toshniwal Paharia, Pooja Toshniwal Paharia. "Review on lateralflow test use spearheaded by SARS-CoV-2 pandemic". NewsMedical. 10 May 2023.

3.

Application Note – Microsphere Coupling Two-step EDC/Sulfo NHS
Covalent Coupling Procedure for Estapor® Carboxyl-modified DyedMicrospheres. MerckMillipore, online.

4.

Application Note – Application Note Performance of Estapor®
Microspheres and Hi-Flow™ Plus Membranes in a Lateral Flow Assayfor Human Chorionic Gonadotropin (hCG). MerckMillipore, online.

5.

Grabarek, Z. and Gergely, J., 1990. Zero-Length Crosslinking
Procedure with the Use of Active Esters. Analytical Biochemistry,185, pp. 131.

6.

Lomant, A. J. and Fairbanks, G., 1976. Chemical Probes ofExtended Biological Structures: Synthesis and Properties ofthe Cleavable Protein Cross-linking Reagent [35S] Dithiobis(succinimidyl propionate). J. Mol. Biol., 104, pp. 243.

7.

Nakajima, N. and Ikada, Y., 1995. Mechanism of Amide
Formation by Carbodiimide for Bioconjugation in Aqueous Media.
Bioconjugate Chemistry, 6(1), pp. 123.

Conjugate pad preparation

8.

33. Prepare conjugate concentration 0.002% or0.005% using conjugate pad buffer.

Staros, J.V., 1988. Membrane-Impermeant Cross-Linking
Reagents: Probes of the Structure and Dynamics of Membrane
Proteins. Accounts of Chemical Research, 21(12), pp. 435.

9.

Staros, J.V., Wright, R.W. and Swingle, D.M., 1986. Enhancement byN-Hydroxysulfosuccinimide of Water-Soluble Carbodiimide-MediatedCoupling Reactions. Analytical Biochemistry, 156, pp. 220.

34. Apply diluted microspheres to 0.7 cm x 30 cm glass
fibre conjugate pad.
35. Dry conjugate pads on fine wire mesh tray
overnight at room temperature.

10. Hermanson, Greg T. 2013 Bioconjugate Techniques, AcademicPress, London.

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