Accepted Manuscript

Title: Capture of red blood cells onto optical sensor for rapid
ABO blood group typing and erythrocyte counting

Authors: Xuemeng Li, Hanbo Feng, Yangyang Wang, Cuiping

Zhou, Wei Jiang, Min Zhong, Jianhua Zhou

PII: S0925-4005(18)30300-9
DOI: https://doi.org/10.1016/j.snb.2018.02.030
Reference: SNB 24125

To appear in: Sensors and Actuators B

Received date: 20-11-2017

Revised date: 31-1-2018
Accepted date: 2-2-2018

Please cite this article as: Xuemeng Li, Hanbo Feng, Yangyang Wang, Cuiping Zhou,
Wei Jiang, Min Zhong, Jianhua Zhou, Capture of red blood cells onto optical sensor
for rapid ABO blood group typing and erythrocyte counting, Sensors and Actuators B:
Chemical https://doi.org/10.1016/j.snb.2018.02.030

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Capture of red blood cells onto optical sensor for rapid ABO
blood group typing and erythrocyte counting

Xuemeng Li a, ǂ, Hanbo Feng a, c, ǂ, Yangyang Wang a, Cuiping Zhou b, Wei Jiang a, Min
Zhong b, Jianhua Zhou a, * zhoujh33@mail.sysu.edu.cn

a
Key Laboratory of Sensing Technology and Biomedical Instruments of Guangdong

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Province, Department of Biomedical Engineering, School of Engineering, Sun Yat-sen

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University, Guangzhou 510006, China
b
Department of Emergency, Nanfang Hospital, Southern Medical University,

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Guangzhou 510515, China
c
Department of Equipment, The First Affiliated Hospital of Guangzhou Medical

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University, Guangzhou 510120, China

*Corresponding

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author: Jianhua Zhou, Tel.: +86 20 39387890; Fax: +86 20 39387890;
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X. M. Li and H. B. Feng contribute equally to this work.
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Highlights:
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 The sensor can perform blood typing and erythrocyte counting simultaneously,
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which has been rarely reported in previous studies.

 The assay method is more sensitive, needs less volume of blood sample and
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requires less time, compared with the common gel microcolumn assay.

 The optical sensing chip is quite promising to achieve full automatization, as

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therefore to save time and avoid human error.

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ABSTRACT

ABO blood group typing and erythrocyte counting are essential for blood
transfusion especially in the vital lifesaving procedures. Here, we developed a simple,
rapid and reliable assay for ABO blood group typing and erythrocyte counting based
on localized surface plasmon resonance biosensor. The optical biosensors were
constructed by the deposition of gold nanoprisms (GNPs) on glass substrates, and the
immobilization of antibodies on the GNPs surface. The sensors were further integrated

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into a microfluidic chip containing two independent sample cells with anti-
isoagglutinins A and anti-B antibodies modified at the bottoms, respectively. The

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sensing chips coupled with ultraviolet-visible (UV-vis) spectrometer were applied for
the identification of blood type A, B, AB and O, respectively; meanwhile, a simple

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proof-of-principle strategy for quantifying red blood cells (RBCs) through the optical

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biosensors were demonstrated with series of RBCs dilution, and observed by UV-vis
spectra. The limit of detection for erythrocyte counting was as low as ~104 cells/mL
(R2=0.99). Additionally, we compared our sensing results with those from conventional

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gel column assay on 7 different blood samples, in which the accuracy of blood typing
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was 100%. The RBC concentrations estimated by our sensor chips were between
3.9~5.0×109 cells/mL, which locates in the normal range of RBCs concentration of
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human. The LSPR biosensors that we developed are capable of qualitative ABO blood
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typing and quantitative erythrocyte counting simultaneously, which have great potential
to develop a preliminary and lab-training-free biomedical device for rapid clinical
diagnosis.
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Keywords: Optical biosensor; Blood typing; Erythrocyte counting; Gold nanoprisms;

Nanoparticle.
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1. Introduction

Blood transfusion is commonly used in multiple clinical settings[1]. Annually,

more than 75 million units of blood are collected and transfused worldwide to supply
for multiple clinical situations in especial the vital lifesaving procedures [2]. However,
it puts forward two crucial issues in blood transfusion. One is the qualitative blood
group typing: blood transfusion without the blood type identification and compatibility
test may cause post-transfusion hemolytic reaction, such as shock, renal failure and
subsequent death [3-5]. Thus, to ensure the safety of transfusion, blood group typing

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must be conducted on all patients. The other significant issue is the quantitative

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erythrocyte counting (i.e. the concentration of red blood cells, RBCs), with a normal
range of 4~6×109 cells/mL [6]. It reflects the actual blood condition of both donors and

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the patients: for blood donors, transfusion should be forbidden if they have anemia; and
for patients, doctors are required to respond quickly to massive hemorrhage or

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accidental trauma during emergency treatment [7-9]. Therefore, an assay to solve both
issues mentioned above, i.e. performing blood typing and erythrocyte counting

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simultaneously, is significant and promising in developing time-saving devices for
clinical application.
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Among those known blood group systems, ABO system is the most common
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applied blood group system, classified by the presence or absence of isoagglutinins A
and/or B on the surfaces of RBCs [10]. By means of identifying the bonding of
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isoagglutinins and corresponding antibody (Ab), several reliable approaches could be

widely used for ABO blood group typing such as conventional slide and tube test,
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microplate slide assay and gel microcolumn assay. However, all as-mentioned
approaches require long performing time, skilled laboratory personnel and specific
laboratory instruments [11]. Alternatively, paper-based and thread-based diagnostic
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assays are promising for prompt and point-of-care (POC) blood group typing, which
are low-cost and convenient but low sensitivity and insufficient accuracy (< 90%) [12-
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14]. As for erythrocyte counting, the automated electrical or optical cell counting
approaches such as the impedance and flow-cytometer, could provide quick and
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accurate processing [15, 16]; while the high-cost, huge-size instruments and high-level
lab training have limited their application in most on-spot diagnosis and primary
hospitals. Therefore, a POC device capable of processing accurate, rapid and
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convenient blood typing and erythrocyte counting is still in need.

Recently, optical biosensor consisting of noble metal (e.g. gold or silver)
nanostructures has been widely applied for powerful quantitative assays, due to its
simplicity, cost-effectiveness [17, 18] and excellent capability for biomedical diagnosis,
such as surface plasmon resonance (SPR), interferometers, waveguides, fiber gratings,
ring resonators, and photonic crystals [17, 19-21]. Optical sensors represent a vital
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classification of analytical tools, and involve a variety of signal transduction pathways
based on absorbance, transmission, fluorescence intensity, refractive index (RI),
polarization and reflectivity etc. [22]. Optical sensing devices, especially in a simple
transmission or reflection mode, are promising for low-cost and mobile healthcare.
Among the numerous optical sensors, the ones based on the localized surface plasmon
resonance (LSPR) of metal nanoparticles have emerged as leading candidates for label-
free, sensitive and specific biosensing [17, 23]. On one hand, the noble metal can be
easily functionalized by some biomolecules (e.g. DNA and antibody) via covalent bond

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such as the Au-S bond to achieve high specificity. On the other hand, the LSPR peak is
able to respond to the RI changes near the nanoparticle’s surface and then the peak shift

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can be utilized as sensitive sensing signal for biodetection [20].
In this study, we developed a prototype of LSPR optical biosensor which could

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simultaneously perform blood group typing and proof-of-principle study of erythrocyte

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counting. The ABO system of blood group typing was involved to demonstrate the
feasibility of this device. The biosensor was constructed by the deposition of gold
nanoprisms (GNPs) on glass substrates and the following immobilization of anti-

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isoagglutinins A/B antibodies (Ab) on the GNPs surface. The as-prepared sensors are
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called as “Ab-GNPs-glass sensors”, and then integrated into a microfluidic chip with
the two sample cells. The blood typing could be determined by capturing the RBCs and
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forming Ab-RBCs complex on the sensing surface in the Ab-GNPs-glass sensor chip
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(Figure 1). By comparing the absorbance changes and LSPR peak shifts of UV-vis
spectra, we could identify the blood type and quantify the concentrations of RBCs as
well. Our Ab-GNPs-glass sensing chip makes it possible to automatically perform the
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ABO blood group typing and erythrocyte counting at the same time on a portable device.
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2. Experiment section

2.1 Materials
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Antibodies against A or B antigens approved for human blood grouping including

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anti-A IgM antibodies and anti-B IgM antibodies (Merck Biopharma, Beijing, China)
were diluted 4 times before use. Polydimethylsiloxane (PDMS, including component A
and B) was bought from Dow Corning, USA. Blood samples were obtained from 7
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adult volunteers with known blood types and provided by the First Affiliated Hospital
of Guangzhou Medical University, China. For all participants, blood types were also
determined with gel microcolumn assay in the center laboratory of the First Affiliated
Hospital of Guangzhou Medical University, China. Samples were stored at 4 °C in
vacuum blood collection tubes containing heparin, citrate and EDTA, and used within
10 days after the collection.
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2.2 Methods

2.2.1 Preparation and characterization of GNPs-glass substrates

The GNPs were prepared in a seed-mediated growth procedure as we previously

described [19]. The product mixture was obtained with expected GNPs and byproducts
such as gold spherical nanoparticles and gold nanorods. The high-yield GNPs were
separated from the mixture by a previously reported procedure [24]. The size and shape
of GNPs (0.1 mg/mL, in aqueous solutions) were characterized with transmission
electron microscopy (TEM, FEI Tecnai G2 Spirit) operated at 120 kV. The GNPs-glass

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substrate was prepared by depositing GNPs onto a glass slide. Briefly, the glass slide

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was cleaned by successively immersed in aqua regia, acetone, ethanol and deionized
water; and then it was dried by nitrogen at room temperature. The GNPs-glass substrate

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was prepared by dripping the GNPs solution (containing ~2 μM

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hexadecyltrimethylammonium bromide) onto the glass slide and then dried. The
morphology of GNPs on the sensor surface was further observed with scanning electron
microscope (SEM, Philips XL-30, Holland). In addition, the ultraviolet-visible (UV-vis)

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spectra of the GNPs-glass substrate were recorded by a UV-2550 UV-vis
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spectrophotometer (Shimadzu, Japan) in the range of 350-900 nm at room temperature.
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2.2.2 Immobilization of blood type antibodies onto GNPs-glass substrates

The GNPs-glass substrates were incubated with 100 μL anti-A or anti-B antibodies
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in phosphate buffer (PBS, pH 7.4) for 2 h, and the antibodies were modified onto the
GNPs via Au-S bond. After incubation, the surfaces of substrates were washed with
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PBS twice and sealed with 3% BSA (w/v) for 0.5 h to prevent nonspecific adsorption.
Subsequently, the substrates were washed with PBS twice to remove the excess BSA
and finally were dried by the nitrogen at room temperature. To measure the variation of
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absorption peak of the substrates, the UV-vis spectra before and after antibody
immobilization were also recorded by the UV-vis spectrophotometer in the range of
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350-900 nm. The sensors functionalizing with antibodies still worked after storing at
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4 °C for 2 weeks.

2.2.3 Fabrication of microfluidic chips integrated with Ab-GNPs-glass sensors

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To integrate anti-A or anti-B Ab-GNPs-glass sensors into a chip, we modified the

two partitioning sides of a GNPs-glass substrate with anti-A or anti-B antibodies
according to the above method. The PDMS mold was made with a puncher to create
two sample cells. After Ab-GNPs-glass sensors were sealed with the PDMS mold, we
obtained the Ab-GNPs-glass senor chip with two sample cells which can detect
isoagglutinins A and B, respectively. The UV-vis spectra of the sensor chips before and
after incubation with different blood types were recorded by the spectrophotometer.
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2.2.4 Measurement of blood type on sensing chips

The RBCs of four types (A, B, AB and O) were prepared by the following method:
the whole blood samples were centrifuged at 1000 rpm for 5 min, and discarded the
supernatant plasma and buffy coat. Then the RBCs pellets were washed with PBS
(pH=7.4) three times. For blood typing, 50 μL 0.3% RBCs suspension (v/v, in PBS,
pH=7.4) of different blood types were dropped into two separate sample cells of the
Ab-GNPs-glass senor chips and incubated for 5 min. After washing with PBS, the
images at 200-fold magnification were taken on an inverted microscopy (Zeiss, German)

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with Axio Observer A1 photographer. The UV-vis absorption spectra were obtained

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before and after the capture of RBCs of four blood types.

2.2.5 Measurement of erythrocyte counting on sensing chips

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To evaluate the detection sensitivity of sensor chips, the concentration of 0.3%

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RBCs (v/v) suspension mentioned above was firstly determined by Beckman cyto
FLEX flow cytometer (Beckman, USA). The suspension of those RBCs was then

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successively diluted to serial concentrations from 102 to 107 cells/mL, in order to
prepare the calibration curve for the erythrocyte counting. The microscopy images and
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UV-vis absorption spectra for different concentrations of RBCs suspension were
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obtained. Each concentration was measured for five times and standard deviations (SDs)
were calculated from those five data points and represented with the error bars. The
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function of absorbance depending on RBCs concentration was determined by non-

linear regression model via software Origin Pro 2016.
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2.2.6 Detection of the blood typing and erythrocyte counting in the real sample by
sensing chips
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To demonstrate the feasibility of our sensor chips, we evaluated their accuracy and
consistency on blood samples in comparison with the commercial method-the gel
microcolumn assay. RBC suspensions (0.3% v/v) of 50 μL prepared as above were
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dropped onto two separate sample cells in the Ab-GNPs-glass sensing chips and
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incubated for 5 min. The UV-vis absorption spectra of the chip after the incubation of
RBCs suspension for different blood samples were obtained. For erythrocyte counting,
the RBC concentration for the real blood was measured for five times and calculated
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by the formula “Conc. =10^ (8.80+2.23×log△Abs.) / 0.3%” with absorbance at 578 nm.

3. Results and discussion

3.1 Fabrication of GNPs-glass substrates and antibodies functionalization

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The LSPR biosensor for blood typing was prepared by the deposition of gold
nanoprisms (GNPs) onto a glass substrate, following by the modification of anti-A or
anti-B antibodies. Figure S1 shows the characteristics of GNPs including the
transmission electron microscope (TEM) image and RI sensitivity of the GNPs. As
shown in the TEM image (Figure S1A), GNPs were in the shape of triangle with small
fillet radius, and the average edge lengths of GNPs were about 100 nm. The
relationship between the LSPR peak shift of GNPs and the RI of the surrounding in
glycerol/water mixtures was shown in Figure S1B. Figure S2A presents a

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representative scanning electron microscope (SEM) image of GNPs on the glass
substrate and shows the overall appearance of the sensor surface. The color of the

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GNPs-glass substrate was dusty blue in naked eyes. The SEM image shows that the
GNPs transformed into circular plates or some irregular-shaped nanostructures on the

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glass substrate. The UV-vis spectra of the GNPs-glass substrate and the GNPs-glass

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substrate modified with antibodies are shown in Figure S2B. The antibodies were
immobilized on GNPs by Au-S bond and acylation. The LSPR peak of the GNPs-glass
substrate was at 677 nm in the visible light region (curve a). After antibody

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functionalization, the LSPR peak moved to 737 nm with a redshift of 60 nm (curve b),
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which indicates the GNPs-glass substrate was successfully modified with antibodies
for capturing RBCs.
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3.2 ABO blood group typing on Ab-GNPs-glass sensing chips

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By measuring the changes of UV-vis absorption spectra, including the

absorbance changes and LSPR peak shifts, the identification of different blood
types (A, B, AB and O) in our experiments were achieved. The results were
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confirmed by the distribution of RBCs on the surfaces of two sensors under

microscopy (Figure 2). Figure 2A reveals the identification result of blood type
A on our Ab-GNPs-glass sensor chip. The spectra of anti-A and anti-B Ab-
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GNPs-glass sensors appeared similar absorption peaks at ~500 nm and ~740

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nm, indicating successful antibodies functionalization on the GNPs-glass

substrate (Figure 2A, curve a and c). After incubating blood type A onto the
anti-A Ab-GNPs-glass sensor, the peak around 740 nm slightly red-shifted and
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additional three absorption peaks appeared around ~420 nm, 545-580 nm

(Figure 2A-Ⅰ, curve b). Three absorption peaks mentioned above are ascribed
to the sole RBCs (Figure S4). The absorbance at the wavelength around 350-
900 nm increased significantly. We inferred that elevating of the entire UV-vis
spectrum was caused by the attachment of RBCs onto the surface of the sensor,
and also by the enhancement of UV-vis absorption induced by LSPR. In Figure
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 2A-Ⅱ, there were almost no difference on the spectra of anti-B Ab-GNPs-glass
sensor before and after the incubation of blood type A. The images of inverted
microscopy were observed that RBCs densely adhered on the anti-A Ab-GNPs-
glass sensor after incubation with blood type A while scarcely on the anti-B
one, which were consistent with changes of UV-vis spectra (Figure 2A, insert).
The results suggest that our Ab-GNPs-glass sensor chip has the ability to
capture RBCs for specific identification of blood types A.

The identification tests were also performed with blood type B (Figure 2B). A

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general elevated UV-vis spectra curve and the presence of RBCs’ characteristic

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absorption peaks around 420 nm and 545-580 nm could be observed for anti-B sensor
(Figure 2B, curve d), while only a low and flat curve appeared on anti-A sensor (Figure

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2B, curve b). These results indicated that the Ab-GNPs-glass sensor chip could
determinate blood type B from the differences in UV-vis spectra. As shown in Figure

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2C, the similar changes of the UV-vis spectra were appeared on both anti-A and anti-B
sensors after incubating the Ab-GNPs-glass sensor chip with blood type AB, i.e. the

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appearance of peaks around 420 nm and 545-580 nm, as well as the general absorbance
increase of entire UV-vis spectra. By contrast, the UV-vis spectra barely changed after
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incubation of blood type O and the adherence of RBCs was hardly observed on both
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anti-A and anti-B sensor surfaces under microscopy (Figure 2D). This phenomenon
indicated our Ab-GNPs-glass sensor chip can be applied for the identification of blood
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type AB and O.

3.3 Erythrocyte counting on Ab-GNPs-glass sensing chips

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The Ab-GNPs-glass sensor chip was also used to estimate the concentrations of
RBCs in blood samples. In order to find the relationship between the concentrations of
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RBCs and the absorbance changes of the UV-vis spectra at a certain wavelength, also
to investigate the sensitivity of the Ab-GNPs-glass sensor, we exposed the sensors to
the diluted RBCs suspensions with serial concentrations from 102 to 107 cells/mL
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(Figure 3). As shown in Figure 3A, the general absorbance of UV-vis spectra gradually
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increased with concentrations of RBCs. The absorbance was clearly boosted in the
range from 104 to 107 cells/mL, while the spectra were hardly distinguished in the lower
concentrations (102-103 cells/mL). We chose the absorbance at 578 nm for further
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analysis, since it was around the wavelengths of the RBCs characteristic peaks, and also
was the proper wavelength for integration into devices. To determine the relationship
between concentrations (Conc.) of RBCs and the absorbance change (△Abs.) at 578 nm,
a formula of non-linear regression fitting was obtained as
[Conc.=10^(8.80+2.23×log△Abs.), R =0.99] was obtained (Figure 3B). Because the
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absorbance change of 0.01 (i.e. 10-2), the reliable readout limit of the spectrometer, was
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corresponding to 104 cells/mL of RBCs, the limit of detection for our sensor was
estimated to be ~104 cells/mL. We also performed a more refined calibration curve in a
narrower range of RBCs from 1.54×106 to 7.7×107 cells/mL, which overlaid the whole
physio-pathological variations in the RBC concentrations (1.2~1.8×107 cells/mL for 0.3%
RBCs suspension, as shown in Figure S5). The amount of RBCs in the visual field of
the microscopy increased gradually with RBCs’ concentrations (Figure 3C), which is
consistent with the results of UV-vis spectra. Since the RBCs of type O have no
isoagglutinins A or B on the cell surface and are unable to do erythrocyte counting in

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the current setting, therefore it is possible to add a third sensor modified with antibody
against common RBCs surface antigen (such as Rh factor or glycophorin A) [8, 25].

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3.4 Consistency evaluation of Ab-GNPs-glass sensor chips

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To verify the reliability of our sensor chips, we compared the results of our assay
and the commercial-available method, i.e. the gel microcolumn assay, which is widely
used in the central laboratories of most hospitals. These comparisons showed that our

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results were in perfect consistence with the gel microcolumn assay on all 7 different
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blood samples and the accuracy was 100% (Table 1). Among the 7 samples, we
estimated the concentrations of RBCs for blood type A, B and AB except the blood
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type O. The concentrations counting by our sensor chips were between 3.9~5.0×109
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cells/mL, which is within the normal range of human RBCs concentration (4~6×109
cells/mL). Compared to the current method of gel microcolumn assay, our senor chip
only requires 5 min and needs less than 10 μL blood sample. Moreover, with the
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absorbance at 578 nm, we could simultaneously estimate the concentration of RBCs by

the above non-linear regression formula, while gel microcolumn assay cannot. The
results suggest that our Ab-GNPs-glass sensor chip is a rapid, simply equipped, semi-
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quantitative blood group identification device.

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4. Conclusions

In this study, we have demonstrated a convenient assay for simultaneous ABO

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typing and erythrocyte counting, using only one sensor chip which was constructed by
GNPs deposition and anti-A/B antibodies modification. The assay method displayed
high accuracy for blood typing, and high sensitivity for erythrocyte counting.
Compared with conventional methods, our sensor is capable of detecting the blood
typing and performing erythrocyte counting simultaneously, which could be beneficial
for rapid evaluation of anemia [4, 11]. This sensing platform has potential for
qualitative and quantitative blood group typing of other blood group systems with
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modification of antibodies against other common RBCs surface antigens on sensing
surface, such as Rh factor. Moreover, our sensor is promising for full automatization,
as therefore to attain time-saving and avoid human error. Our optical biosensor chip is
expected to be used in emergent and resource-limited situations, and the technique has
great potential to apply in the fields of rapid diagnosis, mobile healthcare and public
health security.

Acknowledgments

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We are grateful for the help from Dakewe Biotech Company (Shenzhen). This work
was supported in part by the Ministry of Science and Technology of China

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(2017YFE0102400), National Natural Science Foundation of China (No. 21775168),
Department of Science and Technology of Guangdong Province (2017A020211004),

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and the special support plan for training high-level talents in Guangdong Province (No.
2014TQ01R695). The work was also supported in part by the Australia-China Joint
Institute for Health Technology and Innovation.
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[25] W.L. Then, M.I. Aguilar, G. Garnier,;1; Quantitative blood group typing using surface
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plasmon resonance, Biosens Bioelectron, 73(2015) 79-84.

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Biographies:

Xuemeng Li obtained her Ph.D. degree from Northeast Agriculture University in 2015.
She was a joint Ph.D. student in Faculty of Medical at Helsinki University (Finland).
She currently worked as an associate researcher in Department of Biomedical
Engineering at Sun Yat-sen University. Her current scientific interests are focused on
optical biosensors and its applications in rapid diagnosis and biochemical analysis.

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Hanbo Feng received her Master Degree in biomedical engineering from Sun Yat-sen
University in 2016. She currently works as a clinical engineer in Department of

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Equipment at the First Affiliated Hospital of Guangzhou Medical University.

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Yangyang Wang received her B.S. degree in biomedical engineering from Sun Yat-sen

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University in 2016. She is currently pursuing her M.S. degree in Department of
Biomedical Engineering at Sun Yat-sen University.

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Cuiping Zhou received her M.D. degree from Southern Medical University in 2015.
She currently works as a doctor in Department of Emergency at Nanfang Hospital
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(Guangzhou, China).
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Wei Jiang received his Master Degree in biomedical engineering from Sun Yat-sen
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University in 2016. He currently works as a R. D. Manager in Dakewe Biotech

Company (Shenzhen, China).
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Min Zhong received his M.D. degree from Southern Medical University in 2009. He
currently works as a doctor in Department of Emergency at Nanfang Hospital
(Guangzhou, China).
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Jianhua Zhou obtained his M.Phil. and Ph.D. in chemistry from Tsinghua University
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and the Hong Kong University of Science and Technology, respectively. He was a
Fulbright visiting Ph.D. student studied at the Biomedical Engineering Department of
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Washington University at St. Louis. He is currently an associate professor in the

Department of Biomedical Engineering at Sun Yat-sen University. His research
interests focus on microfluidics and its applications in rapid diagnosis, drug delivery
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and tissue engineering.

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Figures captions
Figure 1. Diagrammatic representation of immobilizing anti-A or B antibodies on gold
nanoprisms (GNPs)-glass substrates and capturing the blood cells upon localized surface
plasmon resonance (LSPR) biosensor surfaces for the ABO blood group typing. Insert:
Anti-A and anti-B Ab-GNPs-glass sensors were integrated into one chip with a
polydimethylsiloxane (PDMS) mold to create two independent sample cells. GNPs: gold
nanoparticles. Ⅰ: anti-A Ab-GNPs-glass sensor; Ⅱ: anti-B Ab-GNPs-glass sensor. The
length of the chip in (A) is 40 mm.

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Figure 2. UV-vis absorption spectra before and after incubation of different blood types-
A, B, AB and O on the surface of anti-A or anti-B Ab-GNPs-glass sensors, respectively.

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(A) blood type A; (B) blood type B; (C) blood type AB; (D) blood type O. Curve a, c were

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measured before incubation of blood；Curve b, d were measured after incubation of blood.
The inserts of each UV-vis absorption spectra are the corresponding microscopy images
after incubation of blood. Ⅰ: anti-A Ab-GNPs-glass sensor；Ⅱ: anti-B Ab-GNPs-glass
sensor.
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Figure 3. Erythrocyte counting using Ab-GNPs-glass sensor chips with serial
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concentrations of RBCs suspensions. (A) UV-Vis absorption spectra after exposure to
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serial concentrations of RBC suspension (0.3%) from blood type A. (B) The relationship
between concentrations (Conc.) of RBCs and absorbance change (△Abs.) at 578 nm. The
error bars in B represent SD calculated from five data points measured at each
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concentration. A formula of the non-linear regression fitting was obtained as

[Conc.=10^(8.80+2.23×log△Abs.), R2=0.99]. (C) Microscopy images of serial
concentrations of the RBCs suspension on the surface of Ab-GNPs-glass sensors, taken
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blood type A and anti-A Ab-GNPs-glass sensor as sample.

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Table
Table 1 Coincidence test of blood group typing between gel microcolumn assay and Ab-
GNPs-glass sensor chips.

Ab-GNPs-glass sensor chips

Gel microcolumn
No. Blood group Erythrocyte counting
assay ΔAbs.
typing (cell/mL)*
1 A A 0.224±0.052 4.7×109 ±1.78×108

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2 A A 0.206±0.071 3.9×109±3.56×108

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3 B B 0.211±0.076 4.1×109±4.15×108
4 O O -

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5 AB AB 0.229±0.058 5.0×109±2.22×108

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6 B B 0.209±0.044 4.1×109±1.20×108
7 O O -

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* The blood samples were prepared to the RBCs suspensions (0.3% v/v) before tests. The
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RBC concentration for the blood samples was measured with in five replications and
calculated by the formula “Conc. =10^ (8.80+2.23×log△Abs.) / 0.3%”.
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