Deliverable 11.3:

Demonstration of Digital immunodiagnostic rapid test (Demo 3)

ML² – Multi Layer Micro Lab

1. Introduction ................................................................................................................................................ 2

1.1. Detection layer ................................................................................................................................... 3

1.2. Layout Design of the Detection layer ................................................................................................. 4

2. Materials and Methods .............................................................................................................................. 5

2.1. Microfluidic immunoassay chip .......................................................................................................... 5

2.2. Fabrication methods: ......................................................................................................................... 6

2.3. Development of the immunoassay for hCG ....................................................................................... 6

2.3.1. Preliminary development of hCG-assayu in 96-microtiter well plate ............................................. 6

2.3.2. Development of fluoroimmunoassay for hCG in microfluidic chip ................................................. 7

2.3.3. Development of the magnetic immunoassay for hCG in microfluidic chip .................................... 7

3. Results ....................................................................................................................................................... 8

3.1. Development of the absorption pad ................................................................................................... 8

3.2. Printing of capture antibodies and magnetic particles ....................................................................... 9

3.3. Magnetic particle flow in the channel together with an applied sample ............................................ 9

3.4. Functionality of the absorption pads ............................................................................................... 10

3.5. Immunoassay development phase: Fluoroimmunoassays ............................................................. 10

3.6. Characterization of the Detection Electronics .................................................................................. 12

3.7. Functionality of the Magnetic Immunoassay ................................................................................... 12

4. Conclusions ............................................................................................................................................. 13

4.1. Conclusions for microfluidics ........................................................................................................... 13

4.2. Conclusions for detection ................................................................................................................ 13

Version Person in charge

v1.0 Leena Hakalahti,

Mika Suhonen

September 15, 2015

1. Introduction

This report 11.3. contains our delivery of the following product:

Demonstration 3: Digital immunodiagnostic rapid test We additionally make recommendations based on our findings for incorporation into future microfluidic-based workflows. We presented the development of the prototyping processes and initial functional test of prototypes for this demonstrator in the reports D8.1. and D8.2.

The primary goal of the MultiLayer-MultiLab (ML2) project Demonstration 3 is to develop a

microfluidics-based rapid immunoassay chip with integrated power supply, transducer readout and display system that is low cost and utilizes high-throughput manufacturing processes. It is based on sandwich immunoassay using superparamagnetic nanoparticles as labels and designed to detect semi-quantitatively analyte concentration. The immunoassay will be demonstrated by measuring hCG (human chorionic gonadotrophin), a hormone released by the placenta right after the embryo begins implanting into the uterine lining. The hormone is released in a pregnant woman's urine. ML

2 draws on the latest advances in

microfluidic based immunoassay.

Figure 1: Illustration of the layered structure of the demonstrator 3. The top layer contains fluidic funtionalities and the bottom layer includes detection coils, read-out electronics and a display.

Figure 2: Schematic picture of the Fluid transport layer. The sample pad collects a sufficient volume of liquid from the urine stream. Then the sample flows into a microfluidic channel. The channel entrance contains a printed conjugate pad that dissolves into the sample liquid. Then sample flows towards the test line and the control line. The antibodies of the test and the control line are located in the detection layer that works as a lid for the channel. The absorption pad maintains the capillary flow in the microfluidic channel.

1.1. Detection layer

The detection layer, microfluidics-based immunoassay chip, contains antibodies of the test and the control line. It also contains magnetic particles with immobilized detection antibodies. The antibodies on the test and the control line are applied by inkjet printing. The inkjet printing utilizes camera registration to ensure accurate placement of antibodies in respect of the detection coils. Superparamagnetic particles are dispensed on the sample inlet area by a suitable dispenser. In this phase the detection coils, read-out electronics and an electrochromic display are located in a separate card.

Figure 3: Model case of Demonstrator 3. Lid with the card containing detection coils, read-out electronics and electrocromic display. Microfluidics-based immunoassay chip is placed on the basesite of the case.The lid is closed during the measurement.

The detection coils are used to detect the presence of captured superparamagnetic particles at the test and the control line. The detection of superparamagnetic particles is based on measuring the resonant frequency of an LC resonator. The LC resonator consists of a planar detection coil and a stable ceramic surface-mount capacitor in parallel. Magnetic particles in the field of the coil affect the inductance of the coil and thus also the resonant frequency of the resonator.

In the development phase Texas Instruments inductance-to-frequency converter LDC1614 is used to measure the resonance frequency. The effective number of bits (ENOB) for the converter is 20 with an approximately 30 ms measuring time per channel. The chip can accommodate four resonance circuits. The current prototype utilizes only two, one is the measurement channel and the other is the reference. The frequency difference between the two resonators depends on the difference of the number of magnetic particles near the measurement coil and the reference coil. Electronics design and measurement principle is presented more detailed in Deliverable 7.1 (VTT).

Figure 4: Detection layer contains detection coils, read-out electronics and a display. The detection coils are covered by a printed dielectric insulator layer, on top of which antibodies of the test and the control line are immobilized.

1.2. Layout Design of the Detection layer

Final design of the printed circuit board (PCB) consists of four conductive layers separated by insulator. The conductive patterns on different layers can be connected with conductive vias. A four-layer design was chosen to ensure signal integrity and noise immunity. An overview of the layout design can be seen in

b)

Figure 5.

a)

b)

Figure 5. a) Board layout overview. Layer 1 (top) is blue, layer 2 pink, layer 3, green and layer 4 (bottom) red. b) Bottom (coil) side of the manufactured PCB.

The detection coils are planar 20-turn square coils. The outside dimensions of the coils are 13.3 mm x 13.3 mm. The conductor width is 0.15 mm and the turns are spaced 0.15 mm apart. There is a 1.5 mm x 1.5 mm empty space in the center of the coils. The coils are on the bottom layer of the PCB. There is one via from the center of the coil and another from a corner of the coil connecting it to other layers.

Figure 6. Detection coil design. The line width and spacing are 0.15 mm and 0.15 mm, respectively.

2. Materials and Methods

2.1. Microfluidic immunoassay chip

Different versions of microfluidic chip were developed and constructed

Microfluidic Chip version 1

bottom: hydrophilic film, 3M MSX-6862, 4 mil ~ 100µm

microfluidic channel tape: double sided, 3M 9965, 50µm thick

lid: 36µm OPET, Luminor

absorption pad: Kromasil 60Å 10µm silica particle + nanocellulose NFC (solid content

1,7%) + deionised water

Microfluidic Chip version 2:

bottom: hydrophilic film, 3M MSX-6862, 4 mil ~ 100µm

microfluidic channel tape: double sided Tesa 220µm thick spacer tape TV01027

lid: 36µm OPET, Luminor

absorption pad: Euca-paper or Technicloth clean room wiping paper, cut with laser into

the right shape

Microfluidic Chip version 3:

Bottom layer: 23µm PET (Mitsubishu Polyester Film, hostaphan RNK), with printed

sample areas of hCG and IgG

Channel layer: two layers of 51 µm double sided tape (3M 9965)

Cover layer: 95µm CA (Clarifoil) with a screen printed suction pad (2 layers of Si 10µm +

PVB 145 + terpionel)

2.2. Fabrication methods:

Microfluidic Chip assembly:

1) Bottom film with adjustment holes, microfluidic channel structures (with adjustment holes) and

lidding tape with adjustment holes and inlet hole were cut with Craft Robo cutter. The

bottomlayer and the channel layer were attached together by feeding through the table top

laminator at room temperature. Magnetic particles were dispensed into the channel with

BioSpot dispenser.

2) In version 1 the absorption pad consisted of Silicaparticles and the mixture for the pad was

dispensed on the pad area by pipetting. It was dried at 60°C for 15 min.

3) In version 3 the screen printed absorption pad consisted of two layers of Si 10µm and PVB

145 + terpionel.

Deposition of biologicals:

Magnetic particle ink: 1mg/mL (7-10x108beads/mL) Dynabeads MyOne Streptavidin T1 +

38µg/mL biotinylated anti-HCG Mab 5014 + 15% saccarose + 0,1% Tween 20 in 12mM PBS pH 7,4.

HCG ink: 0,5mg/mL anti-hCG Mab 5006 + 3% IPA + 0,1% Tween 20 in 12mM PBS pH 7,4 was inkjet printed on 36µm PET substrate which was cleaned with isopropanol and plasma etched with O2 (1 min, 300W) before inkjet printing.

Control ink: 0,5mg/mL mouse IgG + 3% IPA + 0,1% Tween 20 in 12mM PBS pH 7,4.

The antibodies were inkjet printed on the lid bottom surface. After printing the antibodies the lid was laminated together with the channel with tabletop laminator at room temperature.

2.3. Development of the immunoassay for hCG

2.3.1. Preliminary development of hCG-assayu in 96-microtiter well plate

In order to detect the functionality of the components of the hCG-immunoassay the reagents were tested in an assay systems performed in 96-microtiter well plates. The conditions used in the assay are shown below and they were used as a starting point in developing the assay system for the microfluidic chip. Reagents and assay protocol for hCG-assay in microwell plates: hCG-Ag was coated on microwell plates : 0,1µg/well or 0,05µg/well and incubated o/n in +6C, 350rpm. Blocking: 200µl/well with 3 % BSA, RT, 350rpm Washing 2x200µl/well with PBST, 2min, RT, 350rpm 20µl/well biotinylated anti-hCG-magnetic particles + 50µl/well anti-mouse IgG Alexa Fluor 546 (2mg/ml), Inkubated a) 10min, RT, 350rpm ja b) 1h, RT, 350rpm Washing: 3x200µl/well with PBST, 2min, RT, 350rpm Detection of fluorescence intensity in microwell plates was measured by Tecan microwell reader.

2.3.2. Development of fluoroimmunoassay for hCG in microfluidic chip

Performance of the Immunoassay in the microfluidic chip was preliminary tested by using fluorecence labelled anti-hCG antibody

Reagents and assay protocol for hCG-assay in microfluidic chip: Ink formulation: 1mg/ml anti-hCG mab 5006 + 3% IPA + 0,1% Tween 20 in 50mM Na2CO3 pH 9,6 Control: 1mg/ml mouse IgG + 3% IPA + 0,1% Tween 20 in in 50mM Na2CO3 pH 9,6 Substrate: 36µm OPET Channel layer: 220µm douple-sided Tesa, Bottom: Hydrophilic 3M film, Suction Pad: Technicloth

Pre incubation of Ag+Ab: 60µl hCG + 60µl Alexa Fluor-labelled anti-hCG antibody; 15min, RT, 400rpm (hCG-concentration range from 25 to 250 mU/ml) 100 µl of preincubated sample was flown through the microfluidic channel. hCG concentrations used: 500 mIU/mL, 250 mIU/mL, 100 mIU/mL, 50 mIU/mL, 25 mIU/mL and 5 mIU/mL Fluorescence was measured by Typhoon Fluorescence scanner with PMT 500V, resolution 50µm, AF546.

2.3.3. Development of the magnetic immunoassay for hCG in microfluidic chip

Sample preparation:

Sample fluid was bre-incubated (15min, 350rpm) with Superparamagnetic Dynabeads/biotinylated anti-hCG and hCG antigene in PBS. The amount of the magnetic beads in the sample fluid was 3.5-7x10

7

The amount of hCG antigene: 0mIU/ml, 100mIU/ml, 250mIU/ml and 500mIU/ml (three replicates of each concentration)

hCG test in a microfluidic chip – measurements:

hCG0 (0mIU/ml hCG antigene) chips were used as a reference in the measurements

The chip was placed on the setup so that one coil was measuring the IgG control area and the other was measuring the hCG sample area, a glass slide was placed on top of the sample, the given value from the setup was the frequency difference between IgG and hCG sample areas.

In the measurements, the reference (hCG0) was always measured first (approx. 100 measurement points), then (as the measurement was running all the time) it was replaced with the sample (hCG100 and hCG250) for approx. 100 measurement points, .

Averages were calculated from both the reference and the sample values, then they were subtracted and the achieved values are presented

Figure 7. Detection set-up. Detection layer with microfluidic layer on top .

3. Results

Different versions of the microfluidic chip have been constructed according to the description above and in Deliverable 8.1. In regards to sample fluid and magnetic particle flow in channels chip models 1 and 2 were were selected for functionality tests. Further performance of the chips was evaluated with tests that consisted of additional fluid flow tests, tests of the functionality of adsorption pad materials, printing tests for capturing antibodies and printing tests for magnetic particles. For demonstration the performance of the hCG immunoassay was tested in the microfluidic design (one channel) which was adjusted according to the detection layer design (2 coils).

3.1. Development of the absorption pad

Size and shape of the absorption pad suitable for transferring 100 µl sample with sufficient amount of magnetic particles onto the detection area was evaluated. Flow tests were performed in the channels having different dimensions. Channel dimensions varied and were dependent on the assembly process and materials used for the construction. Channel dimensions used in the tests are shown below:

Channel width: 500 µm up to 3 mm

Channel height: 50 µm or 220 µm Coloured water was used as sample liquid Best channel composition resulted in 100 µl of the sample to flow through the channel faster than in 3 minutes. Magnetic particles printed into first part of the channel were flown together with the sample through the channel into the sample pad. In the case when there was no capturing antibody/antigen present in the sample the magnetic particles were drained from the channels into the absorption pad. Flow characteristics of the Chip version 1, containing the particle based absorption pad, was shown to be better. For this phase of the development the particle material was manually dispensed on the adsorption site. Manual dispensing resulted in uneven particle layer and caused unrepeatable sample flow rates. In the next steps dispensing method and the optimization of the composition of the particle suspension will be further studied.

3.2. Printing of capture antibodies and magnetic particles

Capture antibodies and control antibodies were inkjet printed on OPET substrate in the dilution of 0,5mg/mL hCG-antibody in 12mM PBS pH 7,4 containing Isopropanol and Tween 20 with Dimatix ink-jet printer on 36µm PET substrate. The substrate was cleaned and plasma etched with O2 (1 min, 300W) before inkjet printing.

After inkjet printing the PET layer and the channel layer with the bottom layer were laminated together with tabletop laminator at room temperature.

Figure 8. Inkjet printed capture antibodies on laminated OPET film in a microfluidic channel.

Magnetic particle ink was generated by combining 1mg/mL (7-10x108beads/mL) Dynabeads MyOne Streptavidin T1 with 38µg/mL biotinylated anti-HCG Mab 5014 and mixing the particles with 15% saccarose + 0,1% Tween 20 in 12mM PBS pH 7,4.

Magnetic particles were dispensed into the channel with BioSpot dispenser.

Figure 9. Magnetic particles dispensed into a microfluidic channels by BioSpot printer

3.3. Magnetic particle flow in the channel together with an applied sample

Flow of the magnetic particles together with the sample was tested. The magnetic particles were dispensed into the first part of the channel. Results of the magnetic particle flow are shown below in Figure 5.

Figure 10: Dispensed Magnetic particles flow in the channel together with the sample.

3.4. Functionality of the absorption pads

Capillary flow in the channels was tested by using coloured water. Two different type of adsorption pads were both functional and drained the channels in less than 3 minutes.

Figure 11. Chip version 1 (particle based adsorption pad) with 100 µls of coloured water sample absorped into the absorption pad.

Figure 12. Chip version 2 (paper based adsorption pad) with 100 µls of coloured water sample absorped into the absorption pad.

3.5. Immunoassay development phase: Fluoroimmunoassays

In the first testphases the detection was based on fluorescence labelled anti-hCG

antibody. It was shown that biotinylated hCG antibody conjugated with the magnetic particles was immunoactive and suitable for use in the development of microfluidic magnetic particle detection based assays.

Figure 13. hCG-immunoassay in 96-microwell plates. Inkubation times 10 minutes/1 hour

Results obtained for the measurement on hCG-antigen in microfluidic channel. Channel construction and the assay details are described in chapter 2.3.2.

Results are shown below. The results showed the principle conditions of functional hcG-assay in microfluidic channels and thus worked as a intermediate phase in the magnetic assay formulation

Figure 14: hCG-fluoroimmunoassay in microfluidic channels showing fluorescence intensities measured for hCG (400 IU/mL) at the measuring sites of anti-hCG and control IgG as capture antibodies (negative control)

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3.6. Characterization of the Detection Electronics

The prototype was characterized using samples of dried superparamagnetic particles

(Invitrogen Dynabeads MyOne Streptavidin T1 solution on a 20 m thick PET film. The reference frequency level for a resonator was taken to be the resonator frequency when no sample was placed on the resonator. Figure shows the difference of frequency changes of the resonators when different magnetic particle samples were placed on the measurement resonator and a blank sample without magnetic particles on the reference resonator.

Figure 15. Measurement results show the difference in the change of the resonance frequency of the measurement coil and the reference coil when particles are intruduced in the measurement coil. The small nonlinearity of curve may be due to problems in placing and positioning the thin film samples on the coils.

3.7. Functionality of the Magnetic Immunoassay

Magnetic Immunoassay was performed in microfluidic chips accordinf to protocol in 2.3.3.

Measurered results showing a dose-response curve at the range of 0-100 mU/mL hCG. For each concentration the average Δf(Hz) value of three parallel samples is presented in figure 16.

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Figure 16: Dose-response curve for hcG concentration range 0-250 mU/mL.

4. Conclusions

4.1. Conclusions for microfluidics

Fluidic functionality has been tested with different chip layout versions. Structure and functionality of the fluidic layer has been studied thoroughly and the most feasible version has been used in the demonstaration of the magnetic immunoassay in microfluidic channels. Massmanufacturability of the fluidic chip has also been demonstrated. However, there are still some challenges in the fluidic functionality. There are still problems with the continious flow, flow speed and accumulation of airbubbles into the channels. If the chips do not empty properly, some fluid (containing magnetic particles) will be left in the channel thus rresulting in unspecific binding and false measurement results.

Therefore microfluidic development needs to be studied further.

4.2. Conclusions for detection

Electronics for magnetic particle detection was designed and implemented. The new miniaturized prototype was implemented on a convention FR4 PCB. The prototype performs as expected. The coil design is not optimal but may be sufficient for the current application. An implementation on a flexible substrate should be fully possible without any changes to either the circuit design or the layout design although a four-layer flexible process with conductive vias would be required.

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