Electronic Supplementary Material

Article title: Design and development of a microarray processing station (MPS) for automated

miniaturized immunoassays

Journal name: Biomedical Microdevices

Mateu Pla-Roca1*, α, Gizem Altay1*, Xavier Giralt2, Alícia Casals2,4, Josep Samitier1,3,5

1Nanobioengineering Laboratory, Institute for Bioengineering of Catalonia (IBEC), Baldiri

Reixac, 10-12, Barcelona 08028, Spain2Robotics Laboratory, Institute for Bioengineering of Catalonia (IBEC), Baldiri Reixac, 10-12,

Barcelona 08028, Spain3 The Biomedical Research Networking Center in Bioengineering, Biomaterials and

Nanomedicine (CIBER-BBN), Maria de Luna, 11, 50018, Zaragoza, Spain4Center of Research in Biomedical Engineering. Universitat Politècnica de Catalunya, Jordi

Girona, 1-3, 08034 Barcelona5Department of Electronics, University of Barcelona (UB), Martí i Franquès, 1, Barcelona

08028, Spain

*These authors contributed equally to this work.

α Corresponding author contact information:

Nanobioengineering Laboratory, Institute for Bioengineering of Catalonia (IBEC), Baldiri

Reixac, 10-12, Barcelona 08028, Spain

E-mail address: mpla@ibecbarcelona.eu

Tel: +34 93 403 71 38 Fax: +34 93 403 71 38

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1. Microarray processing station general description

The microarray processing station (MPS) is comprised of a washing bowl with apertures,

connections, washing mesa, washing aspiration area with drains, drying nozzles and reagents’

sources (Fig. S1). The MPS can be fixed on a metallic support. Fluids’ (reagents and washing

solutions) and gases’ (for the drying processes) flows are controlled thanks to peristaltic pumps

and solenoid valves’ manifolds. The MPS hardware controlling system actuates the peristaltic

pumps and solenoid valves’ manifolds, and the MPS software allows programming the assay

routines.

Fig. S1 MPS close view.

2. Microarray processing station detailed description of the distinct

components

2.1. Washing bowl: The washing bowl is the recipient where the substrate is placed in order

to perform the automatic washing and drying of the substrate (Fig. S2 a, b). The washing bowl

contains seven wall apertures with internal thread (M3 metric) that allows the screwing of

barbed connectors (for tubing ID: 1/8“) with M3 external thread for easy connection to tubing.

The washing bowl has a washing mesa where the substrate is placed and two waste aspiration

areas with 3 drains each that are perpendicular to the waste aspiration area at the bottom plane

(Figure S2 c). The apertures’ height is adjusted in order to ensure that liquids and gases flow

points the substrate surface. The substrate maximum height that can be used on the current

prototype is adjusted to 1 mm (the standard thickness of microscope glass slides) although can 2/9

be used with thinner substrates. The number of apertures can be easily expanded by simple post-

processing of the washing bowl in order to include more threaded wall apertures. The washing

bowl has been fabricated by FDM 3D printing of acrylonitrile butadiene styrene (ABS)

material. The washing bowl has four neodymium magnets at the bottom that facilitates its

fixation on metallic surfaces (Fig. S2 d).

Fig. S2 The washing bowl (a) side view and (b) plan view showing the apertures and the connectors’

details. (c) MPS section in detail showing the drain channels located on the waste area. (d) Image

showing the neodymium magnets located at the bottom of the MPS that allow fixing it on any metallic

surface.

2.2. Washing Mesa. The central area of the washing bowl is defined as the washing mesa.

The washing mesa (1 mm in height respect to the waste aspiration area) is the area (25 mm x 25

mm) where the substrate (25 mm x 25 mm or less) is positioned (Fig. S3 A, B). The function of

the washing mesa is to ensure a physical separation between the washing mesa and the waste

aspiration area for distinct substrate thickness as is described schematically on Fig. S3 C.

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Fig. S3 Drawing showing the inners part of the MPS. (a) Drawing and (b) image showing in detail the

washing mesa, the waste aspiration area and the drain. (c) Drawing that depicts the washing mesa

functioning as a liquid separator that prevents contaminations from the waste solution.

2.3. Clamping system for fixing the substrate on the washing mesa: The substrate is

fixed on the washing mesa with magnetic clamping (Fig. S4). The magnetic clamping system

consist of an array of eight neodymium cylindrical magnets (Ø = 2 mm, h = 6 mm) inserted on

the washing mesa. Once the substrate is positioned on the washing mesa two or more cylindrical

magnets (Ø = 2 mm, h = 2 mm) magnets can be used to clamp the substrate.

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Fig. S4 Magnetic clamping systems.

2.4. Drying nozzles

Substrate surface is dried with a gas flow. Gas flow is pointed onto the substrate surface thanks

to two metallic nozzles (Fig. S5). The nozzles are connected to the tubing and the gas flow is

controlled with solenoid valves’ manifold.

Fig. S5 Drying nozzles connected to tubing with pressurized gas.

2.5. Peristaltic pumps manifold

The reagents are pumped and the waste solution is aspirated thanks to a manifold of commercial

peristaltic pumps (Kamoer®) (Fig. S6) controlled with the MPS hardware controlling system.

Peristaltic pump tubing (Tygon® tubing, ID1/8”) is connected to the washing bowl using the

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Barbed connection

Tubing for pressurized gas

nozzles

barbed connections. The maximum flow rate that can be achieved with each of peristaltic pump

is 120 mL/min. The peristaltic pumps are based on the step motors which allow a precise

control on the peristaltic pump. Peristaltic pumps tend to induce pulsations on the flow when

operating. This pulse can be used in some cases to induce efficient agitation of the solutions.

Fig. S6 Peristaltic pumps’ manifold.

2.6. Electro valves’ manifold

The electro valves’ manifold (5V actuated solenoid valves from Gems ® normally close type)

open only when actuated, letting the pressurized gas pass through and reach the nozzles. The

gas used for the drying process can be pressurized nitrogen or pressurized air. Gas source can be

either in-line, from a bottle that contains a manometer or from an air compressor (Fig. S7).

Fig. S7 Eletro valves’ (solenoids) manifold.

2.7. MPS controlling hardware

The programming of the rinsing, blocking and drying routines is performed using the MPS

controlling hardware (MCH). The MCH is based on the assembling of the electronic

components all of them commercially available: microprocessor (Arduino Mega®), Arduino

power shield (Sparkfun®), peristaltic pumps drivers (Makerbot®), power supplies (5V and

12V), USB cable connections to the PC and cable connections to peristaltic pumps’ manifold

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Connections to control hardware

Electro valves

Gas nozzles

tubing tubing

pressurized gas source

and electro valves. Microprocessor, power shield and peristaltic pump drivers are open source

components (Fig. S8).

Fig. S8 MPS controlling hardware.

2.8. MPS controlling software: The MPS automated procedures can be easily programmed

creating a .txt file, called step file (Fig. S9). The .txt file with the routine ordered is send to the

Arduino microprocessor programed using C++ language. The automated routines (steps) can be

divided into: (i) system priming (filling of the tubes), (ii) System empting (empting of the tubes)

routines, (iii) wash and dry routines and (iv) reagents loading and (v) mixing routines.

The generated routines are sent to the Arduino microprocessor using a USB cable and

HyperTerminal protocol and COM port for communication.

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Cable connections to electro valves and peristaltic

pumps

USB connection to PC COM port

Microprocessor (Arduino®) and power

shield (Sparkfun®)Peristaltic pump

Drivers (Makerbot®)

Power sources (5 V and 12 V)

Pumps manifold (Kamoer®)

Electro valves manifold (Gems®)

Fig. S9 Programing routines on a .txt file. The routines are sent using SerialComm (HyperTerminal)

communication towards the Arduino microprocessor.

3. Optimization of printing conditions

Working with nL volumes, the amount of material deposited on the surface is also usually very

low. Therefore, in order to obtain good limit of detections it is important to maximize the total

amount of material deposited on the surface. Some important factors that affect the total amount

deposited are the evaporation rate of the sample and the buffer content. The most frequently

used ways to delay the evaporation are the glycerol content and chamber humidity (Wang et al.

2013). Here, these parameters were assessed by printing AF555 IgG (in PBS or PBS_Tween20,

PBST) on nitrocellulose film slides and recording the relative fluorescence intensity (RFI) after

washing and drying the substrate with MPS. It was confirmed that higher the glycerol content

and the humidity better the material adsorption; while the detergent content decreased the total

amount adsorbed (Fig. S10). PBS-10 % Glycerol under 60 % humidity were the optimal

printing conditions used for all the experiments unless stated otherwise.

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Fig. S10 Printing parameters. Graph depicting the effect of glycerol percentage, detergent and humidity

on the amount of protein adsorbed on the nitrocellulose surface. It is seen that higher the glycerol content

and the humidity better the protein adsorption; while the detergent content decreased the total amount

adsorbed.

ESM 2 Video showing MPS installed in microarrayer performing a printing routine

ESM 3 Video showing MPS installed in microarrayer performing a washing routine after

the printing procedure

ESM 4 Video showing the MPS washing and drying the substrate automatically

References

J. Wang, K. Barker, J. Steel, J. Park, J. Saul, F. Festa, G. Wallstrom, X. Yu, X. Bian, K. S.

Anderson, J. D. Figueroa, J. LaBaer, and J. Qiu, PROTEOMICS – Clin. Appl. 7, 378 (2013).

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Humidifier Off (40 %) Humidifier On (60 %) 0

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PBS 0% Glycerol PBS 2% Glycerol PBS 10% Glycerol PBST 0% Glycerol PBST 2% Glycerol PBST 10% Glycerol