Rapid detection and quantification of paper-based

microfluidics using machine learning

Wei Zheng1, Kan Wang1,*, Hao Xu2, Armando Zhu1, Tangan Li1,

Yuemeng Cheng1, Chujun Zheng1, Qihong Ning1, Qinghui Jin3,4, Daxiang

Cui1
1 School of Sensing Science and Engineering, School of Electronic Information and
Electrical Engineering, Shanghai Jiao Tong University, Shanghai Engineering
Research Center for Intelligent diagnosis and treatment instrument, Key Laboratory of
Thin Film and Microfabrication Technology (Ministry of Education), Shanghai 200240,
China.
2School of Naval Architecture, Ocean & Civil Engineering, Shanghai Jiao Tong

University, Shanghai 200240, China

3State Key Laboratory of Transducer Technology, Shanghai Institute of Microsystem

and Information Technology, Chinese Academy of Sciences, Shanghai 200050, China

4Faculty of Electrical Engineering and Computer Science, Ningbo University, Ningbo

315211, China

*Corresponding authors: Kan Wang: wk_xa@163.com

Co-authors Email: Wei Zheng: zheng-wei@sjtu.edu.cn

Hao Xu: xudahao@sjtu.edu.cn
Armando Zhu: armando@sjtu.edu.cn
Tangan Li: andrewl1234@sjtu.edu.cn
Yuemeng Cheng: ym_cheng@sjtu.edu.cn
Chujun Zheng: shulanzcj@sjtu.edu.cn
Qihong Ning: nqihong_a@sjtu.edu.cn
Qinghui Jin: jinqh@mail.sim.ac.cn
Daxiang Cui: dxcui@sjtu.edu.cn

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Abstract

Microfluidic paper-based analytical devices (uPADs) have been widely

used in point-of-care testing owing to their simple operation, low volume

of the sample required, and the lack of the need for an external force. To

obtain accurate semi-quantitative or quantitative results, uPADs need to

respond to the challenges posed by differences in reaction conditions. In

this paper, multi-layer uPADs are fabricated by the imprinting method for

the colorimetric detection of C-Reactive Protein (CRP). Different lighting

conditions and shooting angles of scenes are simulated in image acquisition,

and the detection-related performance of uPADs is improved by using a

machine learning algorithm. The You Only Look Once (YOLO) model is

used to identify the areas of reaction in uPADs. This model can observe an

image only once to predict the objects present in it and their locations. The

YOLO model trained in this study was able to identify all the reaction areas

quickly without incurring any error. These reaction areas were categorized

by classification algorithms to determine the range of risk of CRP

concentration. Multi-layer perceptron, convolutional neural network, and

residual network algorithms were used for the classification tasks, where

the latter yielded the highest accuracy of 96%.

Keywords: uPADs, Machine learning, YOLO, ResNet, C-reactive protein

(CRP)

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1. Introduction

With developments in technologies related to medical science, point-of-

care testing (POCT) [1]-[3] is becoming increasingly popular because of

its portable experimental instruments, simple operation, and instant results.

Paper-based microfluidic analytical devices (uPADs) [4] are widely used

in POCT. They can detect the target analyte in the reaction area of paper-

based devices by constructing specific hydrophilic and hydrophobic

channels for diversion on the paper-based material. Since uPADs were

proposed at the Whitesides Laboratory in 2007 [5], rapid advancements

have been made in research on them recent years. uPADs have been widely

used for disease monitoring, environmental testing, and food quality

testing. Compared with the traditional detection device, it has the

advantages of simple operation, requiring a smaller sample, and not

needing an external force. The methods of detection commonly used in

uPADs include the colorimetry method [6]-[9], electrochemical method

[10]-[12], chemiluminescence method [13], [14], and fluorescence method

[15], [16]. Colorimetry is the most widely used of them. Qualitative results

can be obtained through it by observing whether the reaction area is colored.

However, to obtain accurate semi-quantitative or quantitative results,

uPADs need to respond to the challenges of reagent stability and

differences in lighting conditions.

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 Machine learning is a part of Artificial Intelligence that has been widely

developed in recent years, and has been applied to a number of areas,

including biomedicine [17]-[21]. Machine learning does not increase the

cost of hardware or the complexity of the detection program used while

improving the detection performance of the equipment. This suggests that

it can improve the detection performance of uPADs as well. The

application of machine learning to biomedicine has focused primarily on

images. Machine learning algorithms have been used for such a variety of

tasks as object detection [22], [23], image segmentation [24]-[26], and

image classification [27]. Machine learning algorithms accomplish these

tasks by training models to identify complex nonlinear patterns used as

input. Machine learning algorithm can be used in uPADs to detect targets

in the reaction area. The reaction area is further analyzed to obtain more

accurate results. Compared with traditional image processing algorithms,

an increase in sample size can more significantly improve the performance

of machine learning algorithms without the need for complex programs. In

addition, the diversity of training samples can enhance the robustness of

the machine learning model. Further, when the sample is weakly positive

and the signal of the reaction area is weak, its color is close to that of the

background. Conventional threshold segmentation uses only the color

feature, which cannot adequately identify the boundary of the reaction area,

and thus misses it. The feature learning characteristic of machine learning

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methods can solve this problem.

As a typical inflammatory biomarker, the C- reactive protein (CRP) can

be used to screen the grade of inflammation and identify the existence of

acute organic diseases, and to diagnose related diseases [28]-[31]. The CRP

level in blood is an indicator of atherosclerosis, and is known to be a

predictor of heart attack and stroke. CRP detection is quantitative detection

commonly required by cardiologists. According to the clinical definition

of its cut-off value, patients are divided into low-, medium-, and high-risk

groups: lower than 1 ug/mL is considered to be low risk, 1–3 ug/mL is

medium risk, and higher than 3 ug/mL is high risk. Therefore, it is

important to detect the concentration of CRP and correctly classify the

range of risk of CRP concentration in patients with heart disease.

In this study, the CRP concentration is detected by combining uPADs

with machine learning algorithms. Custom-designed uPADs for CRP

detection are prepared by using the imprinting method. The object

detection model [32] in machine learning is used to accurately detect the

reaction areas of uPADs. Following their recognition, different machine

learning algorithms, including the multi-layer perceptron (MLP),

convolutional neural network (CNN), and residual network (ResNet) [33],

are used to classify the reaction areas and determine the range of risk of

CRP concentration in them. The contributions of this work include using

machine learning for object detection in the reaction areas of uPADs,

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collecting images of uPADs under different illumination conditions and tilt

angles to simulate empirically acquired scenes, and enhance the robustness

of the detection model, using different machine learning algorithms to

classify the reaction areas under different conditions, and comparing the

results of the classifications. At the same time, the classification model

combines the reagent subpackage batch information and uPADs

manufacturing batch information to further improve the detection effect of

uPADs. ResNet was found to have the highest accuracy.

2. Experiments

2.1 Materials and instruments

Mouse monoclonal CRP coating antibody, mouse monoclonal CRP

labeling antibody conjugated with horseradish peroxidase (HRP), and

purified standard solution of human CRP (Linc-Bio Science Co., Ltd.,

China). Whatman grade-1 chromatography paper (GE Healthcare Ltd.).

Chitosan powder (Sinopharm Chemical Reagent Co., Ltd., China).

Glutaraldehyde (Aladdin Chemical Reagent Co., Ltd., China). Bovine

serum albumin (BSA, standard grade) (MP Biomedicals LLC, USA).

One-step 3,3’,5,5’-tetramethylbenzidine (TMB) chromogen solution

(Beyotime Biotechnology Co., Ltd., China). Hexane solution (Macklin

Biochemical Co., Ltd., China), triethanolamine (Shanghai Yien Chemical

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Technology Co., Ltd., China), and ethanol (Aladdin Chemical Reagent Co.,

Ltd., China). The blocking buffer comprised 1× phosphate-buffered saline

(PBS, pH 7.4) containing 0.5% (w/v) BSA. The washing buffer comprised

0.05% (v/v) Tween-20 in 1× PBS (pH 7.4). Ultrapure water with a

resistivity of 18.2 MΩ cm (25 °C) was used to prepare the solutions in all

steps.

The contact angle was measured with a drop shape analyzer (DSA100,

KRÜSS GmbH, Germany). A Xiaomi 8 smartphone was selected as the

tool for image acquisition. A custom-designed 3D-printed uPAD carrier

was fabricated by WeNext Technology Co., Ltd. (Guangdong, China), and

a custom-designed photosensitive stamp was bought from a local stamp

shop.

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2.2 Design and fabrication of detection platform

2.2.1 Design of detection platform

Fig. 1 The detection platform. A. Two-layered uPAD. B. The uPAD during the
reaction. C. The uPAD during washing. D. 3D-printed carrier. E. 3D-printed carrier
with uPAD.

The designed detection platform consisted of two parts, a two-layered

uPAD and a 3D-printed carrier. As shown in Fig. 1A, the two-layered

uPAD comprised a top washing layer and a detection layer. The washing

layer contained a central circular sampling area 6 mm in diameter with five

microchannels (3 mm × 8 mm), which radiated uniformly from the central

sampling area. The size of the detection layer was 4 cm × 4 cm, and it

contained five circular reaction areas, each of which was 4 mm in diameter.

The detection layer is hydrophobic except at the five reaction areas. The

sampling and reaction areas were circular so that the solution could flow

uniformly. In the washing steps, the washing layer was appropriately

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placed so that the five microchannels could come into contact with the five

reaction areas of the detection layer. Dropping the washing buffer into the

sampling area can enable it to reach the reaction areas through the

microchannels and wash them. Except during washing, the washing layer

prevented contact between the microchannels and the reaction areas by

rotating at an appropriate angle (Figs. 1B, C).

To avoid interference due to the contact between the back of the

detection layer and the environment during the reaction, a 3D-printed

carrier was designed and manufactured to carry the uPAD. The 3D-printed

carrier was a hollow cube with an external length of 4.2 cm and internal

length of 3.8 cm, with a 2 mm depression inside. Photographs of the 3D-

printed carrier and the uPAD on it are shown in Figs. 1D and E,

respectively. To simulate different inclination angles when taking the

photos, brackets with inclination angles of 15° and 45° were also fabricated

for image acquisition.

2.2.2 Fabrication of detection platform

The wax printing method [34] is the most widely used to fabricate uPADs

[35]. However, it requires a professional wax printer or a modified ordinary

printer. The imprinting method [36] is a good alternative, and has the

advantages of a simple operation and low cost. Various

hydrophilic\hydrophobic channel patterns can be designed and the

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Fig. 2 Imprinting method for preparing uPADs. A. Photosensitive stamp pad. B.
Custom-designed photosensitive stamp pad with hydrophilic channels. C.
Photosensitive stamp pad soaked with blocking agent. D. Photosensitive stamp. E.
Filter paper (4 cm × 4 cm). F. Filter paper soaked with AKD solution. G. Hydrophobic
filter paper imprinted by blocking agent. H. Prepared uPAD.

corresponding photosensitive stamp made that are then printed on the

surface of the filter paper. The steps of preparing uPADs using the

imprinting method are shown in Fig. 2. First, we cut the Whatman grade-1

chromatography paper into an appropriate size (4 cm × 4 cm), and soaked

it into a hexane solution containing an alkylketone dimmer (AKD) for 5

minutes for its hydrophobization. Second, we use the pattern designed to

prepare the photosensitive stamp to fill it with the triethanolamine solution

as blocking agent. Lastly, we imprinted the blocking agent on the

hydrophobic filter paper using the prepared stamp and heated it at 105 °C

oven for 5 minutes. Following the above steps, the non-imprinted area was

hydrophobic and the imprinted area was hydrophilic. uPADs with specific

hydrophilic\hydrophobic channels were hence prepared.

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2.3 Experimental operation of CRP detection

The processes of modification and sandwich immunoassay of CRP are as

follows. A total of 4.0 μL 0.5 mg/mL chitosan (0.05%, V/V) was applied

to the reaction area and then dried at room temperature for 15 minutes.

Because of the opposite charges, cationic chitosan remained on the surface

of the anionic cellulose reaction area. Following this, 4.0 μL of a 0.25%

(V/V) glutaraldehyde solution was added to react with the chitosan for 2 h.

The aldehyde group of glutaraldehyde was exposed on the surface of the

reaction zone to covalently fix the coating antibody. A total of 4.0 μL of

45 μg/mL mouse monoclonal CRP coating as antibody was incubated on

the reaction area for 15 minutes. Then, we dropped a 4.0 μL blocking buffer

to block the reaction area for 15 minutes to occupy any unbound sites on

the paper and avoid non-specific binding. Washing was performed three

times using the washing buffer. Next, 4.0 μL of the CRP at different

concentrations was added to each reaction area for 10 minutes, and each

area was washed three times. Then, 4.0 μL of 1.43 μg/mL HRP antibody

was added to the reaction areas for 5 minutes. Washing was then performed

five times. Finally, 4.0 μL of the TMB substrate solution was added to each

reaction area and reacted for 3 min. The color signals generated on the

surface of the reaction area were analyzed.

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 After the reaction, images of the uPADs were collected by the

smartphone (XiaoMi 8) for subsequent network training. To simulate the

scene of detection and increase the robustness of the model, images were

taken under three illumination conditions: natural light, indoor light, and

flash light. The tilt angle of the uPADs during image acquisition was also

changed. The images were taken without tilt, and with tilts of 15° and 45°

by placing uPADs on the brackets at the corresponding inclination angle.

Finally, 98 images of the uPADs have been collected. Since each uPAD

has five reaction areas, a total of 490 reaction area images can be obtained.

Training set and testing set were prepared using k-fold cross-validation.

2.4 YOLO to detect reaction areas

When uPADs were used for POCT, various image acquisition

environments with different lighting conditions were considered. Images

were also taken manually, and a fixed shooting angle was not guaranteed.

Therefore, traditional image processing algorithms face the following

challenges to identify the reaction areas of uPADs: First, the lighting

conditions in different environments require tuning different parameters

for the algorithm. Second, when photos are taken, certain inclination angles

may cause the algorithm to fail. Third, when the sample is weakly positive,

the background color of the detection layer is similar to the reaction area,

which may lead to the omission of the reaction area. Machine learning

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algorithms are expected to solve these problems in traditional image

processing algorithms.

2.4.1 YOLO structure

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Many researchers have proposed various algorithms based on machine

learning for image classification and object detection. The reaction area of

μPADs can be detected by using object detection algorithms as well. The

You Only Look Once (YOLO) algorithm is a widely used one-stage object

detection algorithm that frames object detection as a regression problem to

spatially separated bounding boxes and the associated class probabilities.

Because the entire detection pipeline is a single network, it can be

optimized directly in an end-to-end manner to improve detection.

Compared with other target detection algorithms, YOLO requires fewer

Fig. 3 The YOLOv3 structure for detecting reaction areas of the uPADs.

samples to complete the training of the model, which makes it suitable for

object detection in the reaction areas of uPADs. We used YOLOv3 [27]

to detect the reaction areas of the μPADs and the network structure is

shown in Fig.3.

The input to the YOLOv3 is an entire given image, and the network

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outputs vectors providing information on the bounding boxes, class

predictions and, predictions of objects. The network predicts four

coordinates for each bounding box: tx, ty, tw, and th. If the cell is offset

from the top-left corner of the image by (cx, cy), and the bounding box

prior has width and height pw and ph, respectively, YOLOv3 predicts an

“objectness score” for each bounding box by using logistic regression. If a

bounding box prior is not assigned to a ground truth object, it incurs no

loss in coordinate or class prediction, and does so only in terms of

objectness. YOLOv3 uses independent logistic classifiers to predict the

classes for each bounding box. It uses a network structure called darknet-

53 (containing 53 convolution layers) for basic image feature extraction. It

draws lessons from the residual network and sets up shortcut connections

between some layers. To detect objects of different sizes, YOLOv3 outputs

three feature maps in total and predicts boxes at three scales. K-means

clustering was used to determine the bounding box priors. The network

arbitrarily chose nine clusters and three scales, and then divided them up

evenly across the scales.

2.4.2 Label annotation and important training parameters

As described in Section 2.3, a large number of custom-designed μPADs

were prepared in batches using the imprinting method and their images

were captured by the Xiaomi 8 smartphone. The collected images were

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marked using LabelImg software, the reaction areas were framed with

rectangular frames. XML files containing the locations of the reaction

areas were generated and were converted to TXT files. The labeled images

were input to the YOLO network for training. The appropriate parameters

can cause the network to converge more quickly and can reduce its

complexity. The crucial parameters selected in training were as follows.

The images were resized to 640 × 640 to input to the network. The number

of training epochs was 100 and the batch size was 32. The one-cycle

learning rate scheduling strategy was used, with an initial learning rate of

0.01 and a final learning rate of 0.002 using the Stochastic Gradient

Descent optimizer. Three warm-up epochs were used, the warm-up

momentum was 0.8, and warm-up bias learning rate was 0.1. The

momentum and weight decay were, respectively, set to 0.937 and 0.0005.

After training, the network could automatically identify the reaction areas

of uPADs.

2.5 Machine learning methods for classification of CRP

concentration

We used the trained YOLO model to identify the reaction areas in the

images. These areas were then classified by using different classification

algorithms to determine the range of risk of CRP concentration. Three

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classification models were selected for image classification: the MLP,

CNN, and ResNet. They are widely used classification algorithms in

machine learning. MLP completes a classification task by using manually

extracted features, and CNN and ResNet do so by directly using images as

input. MLP is a classical neural network model that can deal with non-

linearly separable problems. By manually extracting features as the input

to the network and learning through the hidden layer, the classification task

is completed in the output layer. In this study, the color information of the

identified reaction area was used as the feature input, and the RGB color

space and HSV color space were used. Their mean values were taken and

normalized to form the input vector. After learning in the hidden layer, the

model predicted the range of risk of CRP concentration in the output layer.

In addition, the reagent batch ID and fabrication batch ID were combined

as feature inputs to train the network to improve the results of classification.

The CNN directly used the image as input, and used the convolution layer

and the pooling layer as middle layers to train the network. The identified

image of the reaction area was directly used as the input to the network,

and the classification task was carried out through network self-learning.

We used a simple CNN network to classify CRP concentration. ResNet [28]

is a variant of the CNN that uses residual units.

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3. Results and discussion

3.1 Evaluation of uPADs

We prepared uPADs with excellent hydrophilic and hydrophobic effects

by the imprinting method. Different concentrations of AKD in the

hydrophobic agent have a significant impact on the hydrophilic and

hydrophobic effects of uPADs. With the increase in AKD concentration,

the hydrophobicity of the filter paper increased gradually. When the

concentration of AKD was low, the hydrophobic area of the filter paper

has poor ability to limit the aqueous solution, and the liquid diffused. When

the concentration of AKD was high, the hydrophobicity of the imprinted

area was too high, penetration by the liquid was slow, and it could not fill

the imprinted area. Therefore, we needed to select an appropriate

concentration of AKD to ensure that the uPADs had good hydrophilic and

hydrophobic effects. After optimization, when the concentration of AKD

was 3.0 g/L, satisfactory hydrophilic and hydrophobic effects were

obtained.

In addition, the selection of the blocking reagent has an impact on the

hydrophilicity and hydrophobicity of uPADs. A suitable range of pH for

the reaction between AKD and cellulose is 7.5~8.5. Acids, alkalis, oxidants,

and surfactants can prevent the reaction. Triethanolamine has a good

blocking effect and is highly viscous, where this is suitable for imprinting.

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Triethanolamine was thus selected as blocking reagent. Triethanolamine

has extremely high viscosity, and the photosensitive stamp absorbed it

slowly. When the triethanolamine solution was directly imprinted, it left a

large amount of residue in the imprinted area that affected the subsequent

analysis. Thus, it needed to be properly diluted. When ethanol was used as

diluent, the viscosity of the solution was low when the ratio of

triethanolamine was low, diffusion was prominent after imprinting, and the

hydrophilicity of the imprinted area was low. When the volume ratio of

triethanolamine to absolute ethanol was 1:1, a satisfactory effect was

achieved.

As shown in Fig. 4, the contact angles of the hydrophobic regions were

134.8° and 135.3°, and those of hydrophilic regions were 20.1° and 22.8°.

This indicates that the uPADs prepared by the imprinting method yielded

good hydrophilic/hydrophobic effects.

Fig. 4 Contact angles of hydrophobic and hydrophilic regions.

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3.2 CRP detection performance

The CRP detection experiment was carried out on the uPADs by using

colorimetry. The concentrations of the coating antibody (C-Ab) and HRP

labeled antibody (HRP-Ab) were 45 ug/mL and 1.43 ug/mL, respectively.

Different concentrations of standard antigen solutions were prepared with

a 3 mg/mL CRP antigen solution using PBS. After the reaction, the surface

of the final reaction area was blue, and it was photographed. The higher

the concentration of the CRP antigen was, the darker the blue in the

reaction area was.

In the entire reaction process, the number of applications of washing

needed to be set appropriately. If the samples were not washed enough

times, non-specific adsorption was not fully flushed. Conversely, excessive

washing led to more, cumbersome experimental steps and longer detection

time, and might have destroyed the hydrophilicity and hydrophobicity of

uPADs. Through optimization, the number of washing times of the three

washing procedures are set to 3, 3 and 5 respectively. Similarly, if the

reaction time was too short, the corresponding reaction (surface

modification reaction, antibody coating reaction, BSA blocking reaction,

CRP sandwich immunoassay reaction and enzyme reaction) was

insufficient, and if it was too long, the time needed for detection increased,

and might have led to an excessive reaction. We set the reaction time of

the antigen to 10 min, that of HRP-Ab to 5 min, that of TMB to 3 min, and

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that of the other reagents to 15 min. These reaction conditions yielded

uPADs with excellent color reaction (Fig. 5).

Fig. 5 Photograph of the uPAD used for CRP detection.

3.3 Results of YOLO model for detecting reaction areas of uPADs

The biggest challenge encountered by traditional image processing

methods in recognizing the reaction areas of uPADs is that when the signal

is weakly positive, the color of the reaction area is light, and is similar to

that of the background area. This leads to an inability to identify the

boundary of the reaction area during threshold segmentation, which leads

to its omission. In addition, because the collected images are captured

manually and the image acquisition environment involves different

lighting conditions, traditional image processing methods need different

configurations for different application scenarios. As shown in Fig. 6,

different threshold segmentation methods have different effects. Only by

manually adjusting the threshold to a certain range can the reaction area of

the paper-based microfluidic device be preserved. For different lighting

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conditions, this range of threshold changes. However, it is impossible for

each user to write corresponding programs, which makes it difficult to use

traditional image processing methods to the POCT of uPADs.

Fig. 6 Different uPAD images processed with different threshold methods. A.

Original image. B. Gray image. C. Adaptive mean threshold image. D. Adaptive
Gaussian threshold image. E. Global threshold image. F~K. Global threshold image
with different thresholds (80, 110, 127, 150, 180, 200).

Machine learning methods can solve the above problems. The YOLO

model needs to only label the collected images and use them in the network

for training. The model can learn by supervision, and generates the

corresponding network for object detection. The images of the uPADs

were first collected by smartphone using its flashlight function and were

used for network training. All the reaction areas of uPADs in the test set

were successfully detected. This proves the feasibility of the YOLO model

for the detection of the reaction areas of uPADs. To simulate empirical

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lighting conditions, image acquisition was also carried out with indoor

light and natural light. To increase the robustness of the model, the images

of the uPADs were collected at tilting angles of 15° and 45°. Some images

were used as training sets and the rest were used as the test set. All reaction

areas of the uPADs under different conditions were successfully detected.

The training performance of YOLO is shown in Fig. 7. It can be seen from

Fig. 7. A, B that the bounding box losses of both the training set and testing

set decrease consistently. The minimal gap between training loss and

testing loss indicates that the model has good generalization ability and is

not over-fitting. The recall and mean average precision (Fig. 7. C, D) of

the trained YOLO model are close to 1, demonstrating excellent

performance in reaction area detection. It reflects the significant

advantages and expansibility of the machine learning method. In case of

uPADs with different structures, models that deliver excellent detection

can be generated through network training.

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Fig. 7 Training performance of YOLO. A. Bounding box loss in training set. B.
Bounding box loss in testing set. C. Recall of trained YOLO model. D. Mean average
precision of trained YOLO model. The abscissa of each figure indicates the training
epochs.

3.4 Results of three classification models

After the image of uPAD is input into the YOLO model, the location of the

reaction areas of uPAD will be obtained. According to the location

information provided, part of the reaction areas of the input image were

cropped. Machine learning-based classification models were then used to

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classify the identified reaction areas to determine the range of risk of CRP

concentration. Three machine learning models—MLP, CNN, and

ResNet—were used to classify CRP concentration according to the images

of the reaction areas under different lighting conditions and tilt angles. The

classification of CRP concentration using MLP requires artificial feature

selection. The color features of the RGB color space and HSV color space

were extracted as the input features for the training network, and the final

classification accuracy was 80%. The results of classification were not

satisfactory because even if the color signals of the reaction areas were the

same, data on the color space were different under different lighting

conditions. In addition, we did not use industry-grade equipment during

the preparation and reaction of the uPADs to control the temperature and

humidity of the environment, and several experimental steps involved

manual operation that led to variations in fabrication and operation. The

reagents prepared in different batches also introduced the problem of

reagent stability. We introduced the reagent batch ID and fabrication batch

ID as input features for network training, and this improved the final

training accuracy to 85%. Because the CNN network uses the entire image

as input, there was no need to manually extract features, which led to a

simpler process. Its classification accuracy was 91%. It was not sufficiently

high because the network was not deep enough, and learned few feature

dimensions. ResNet has deeper layers and uses residual units. Its final

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classification accuracy was 96%. Some classification indicators (accuracy,

recall, and F1 score) of the three models are listed in Table 1.

Table 1 Classification indicators of the three model.

Indicator
Precision Recall F1
CRP

MLP 85.23% 76.53% 80.65%

Low
CNN 96.51% 84.69% 90.21%
(0~1 ug/mL)
ResNet 96.91% 95.92% 96.41%

MLP 77.48% 87.76% 82.30%

Medium
CNN 87.25% 90.82% 89.00%
(1~3 ug/mL)
ResNet 93.63% 97.45% 95.50%

MLP 93.89% 86.22% 89.89%

High
CNN 92.50% 94.39% 93.44%
(3~10 ug/mL)
ResNet 98.94% 95.41% 97.14%

As can be seen from the data of Table 1, ResNet performs best in all

indicators. Confusion matrices were also used to evaluate the classification

accuracy of the three models (Fig. 8). It shows that ResNet had the best

classification effect, which is consistent with its accuracy score and other

three indicators. In addition, no incorrect classification occurred between

the low and high ranges of concentration of the three models, indicating

their reliability.

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Fig. 8 Confusion matrixes of three machine learning models.

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4. Conclusion

In this work, we used machine learning methods to detect reaction areas in

uPADs and classified them to determine the risk of heart disease. The

imprinting method yielded uPADs with excellent hydrophilic and

hydrophobic effects, and the immunoassay of CRP was performed on the

prepared uPADs. After the reaction, images were captured under different

lighting conditions and tilt angles to simulate the real detection scene. The

YOLOv3 algorithm was used to identify the reaction areas of the uPAD.

The trained YOLO model yielded perfect detection, and could identify all

reaction areas under different photographing conditions. Following this,

three classification models (ResNet, CNN, and MLP) were used to classify

the detected reaction areas to determine the risk of heart disease. ResNet

achieved the best classification performance, with an accuracy of 96%.

This method is portable, and can be extended to detect other biomolecules

by changing the reagents used. When the shape of the reaction area of

uPADs is different, there is no need to rewrite the program. The recognition

and classification tasks can be accomplished by manually marking the

reaction areas for network training. In addition, image acquisition in this

method simulates the real detection scene. The algorithm delivers good

recognition performance and classification accuracy under different

photographing conditions.

Electronic copy available at: https://ssrn.com/abstract=3989551

Declaration of Competing Interest

The authors declare that they have no known competing financial

interests or personal relationships that could have appeared to influence

the work reported in this paper.

Acknowledgments

We are grateful for the financial support by the National Key Research and

Development Program of China (Grant Nos. 2017FYA0205303 and

2017FYA0205301), the National Natural Science Foundation of China

(Grant No. 32171373), and the Medical Engineering Cross Project of SJTU

(Nos. ZH2018QNA03，YG2019QNB09 and YG2021QN141).

Electronic copy available at: https://ssrn.com/abstract=3989551

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Electronic copy available at: https://ssrn.com/abstract=3989551

Rapid detection and quantification of paper-based
microfluidics using machine learning

Wei Zheng1, Kan Wang1,*, Hao Xu2, Armando Zhu1, Tangan Li1,

Yuemeng Cheng1, Chujun Zheng1, Qihong Ning1, Qinghui Jin3,4, Daxiang

Cui1
1 School of Sensing Science and Engineering, School of Electronic Information and
Electrical Engineering, Shanghai Jiao Tong University, Shanghai Engineering
Research Center for Intelligent diagnosis and treatment instrument, Key Laboratory of
Thin Film and Microfabrication Technology (Ministry of Education), Shanghai 200240,
China.
2School of Naval Architecture, Ocean & Civil Engineering, Shanghai Jiao Tong

University, Shanghai 200240, China

3State Key Laboratory of Transducer Technology, Shanghai Institute of Microsystem

and Information Technology, Chinese Academy of Sciences, Shanghai 200050, China

4Faculty of Electrical Engineering and Computer Science, Ningbo University, Ningbo

315211, China

*Corresponding authors: Kan Wang: wk_xa@163.com

Co-authors Email: Wei Zheng: zheng-wei@sjtu.edu.cn

Hao Xu: xudahao@sjtu.edu.cn
Armando Zhu: armando@sjtu.edu.cn
Tangan Li: andrewl1234@sjtu.edu.cn
Yuemeng Cheng: ym_cheng@sjtu.edu.cn
Chujun Zheng: shulanzcj@sjtu.edu.cn
Qihong Ning: nqihong_a@sjtu.edu.cn
Qinghui Jin: jinqh@mail.sim.ac.cn
Daxiang Cui: dxcui@sjtu.edu.cn

Electronic copy available at: https://ssrn.com/abstract=3989551