Microfluidic Platform for Aptamer based Fluorimetric Analysis of

Analytes

Tanu Bhardwaj and Sandeep Kumar Jha*

Centre for Biomedical Engineering, Indian Institute of Technology Delhi, New Delhi-110016, India

Keywords: PMMA, Microfluidics, Aptamer, Biosensing, FAM, Fluorescence.

Abstract: In this work, we are reporting fabrication of a simple and low cost setup for fluorescence detection based on

aptamer probes. For this reason, we fabricated a PMMA-PMMA microfluidic chip using easily available

laboratory techniques and combined the chip with a simple fluorescence detection setup using optical fiber,

filter, detector and a commercial spectroscopy software. In this new approach, we used two different

strategies to use aptamers as probe. In first strategy, detection of any nucleic acid could be targeted using

simple DNA hybridization with aptamer probe. Such strategy can be used in analysis of samples with

specific nucleic acid sequence, such as pathogen. We proved this using known sequence of ssDNA aptamer

probe immobilized on detection zone on microchip and its FAM labeled complementary strand was passed

over it using microfluidic condition. In other strategy, we attempted detection of any protein or biomarker

using sandwich fluorimetric technique with primary and labeled secondary aptamer immobilized on sensing

region. For this, we used thrombin as model target to validate our setup. Both the strategies proved

satisfactory on our setup. Even more, LOD was also impressive. In future, this setup could further be

miniaturized by using a small on-chip CCD array detector, microcontroller based electronics and LabVIEW

software based control.

1 INTRODUCTION

Aptamers are single stranded nucleic acids, DNA or

RNA, which selectively and specifically bind to a

target molecule such as nucleic acids, proteins, cells,

microorganisms, etc. interacting via weak molecular

forces. They often complement antibodies in terms

of binding efficiency and specificity, yet are more

stable and cheaper to produce. Aptamers are picked

for a particular target from a process called SELEX

(Systematic evolution of ligands by exponential

enrichment) which is comparatively simple process

compared to typical antibody production using

hybridoma technique. In addition, host animal is also

not required for the production of aptamers. Extra

resistance against denaturation and ease of chemical

modifications make aptamers friendlier to use in

biosensing (Tennico, 2010; Song, 2012).

Meanwhile, microfluidics has replaced many

analytical and biomedical techniques due to its

reduced size, cost and reagents utilization. Aptamers

and their application in microfluidics are inspiring

researchers to create a bridge between two

drastically different fields of biological and

analytical techniques. Aptamers with microfluidics

have already been used for analysis of biological

targets/analytes like thrombin, VEG-165, peptides,

cancer cells, C-reactive protein, viruses, microbes

and various other proteins or biomarkers (Xu, 2010).

Out of which, viruses, pathogens or microbes are

identified by these aptamers due to their specific

microbial proteins, lipopolysaccharides or nucleic

acids. On the other hand, biomarkers (proteins)

originating in the case of cancer allow their detection

using aptamers (Viscidi, 1987; Su, 2015).

Detection of various diseases associated with

microbes was made possible by simple hybridization

of aptamer probe labelled with dye to the

complementary nucleic acid strand from the microbe

(Tennico, 2010). Besides, aptamers and

hybridization principle has been used for DNA

microarrays, single nucleotide polymorphism

detection, gene expression studies and nucleic acid

diagnostic applications (Wang, 2011; Abu-Salah,

2015). Furthermore, various proteins and biomarkers

have been identified using primary and dye labeled

secondary aptamers sandwich assay like ELISA

technique. The use of fluorescent dyes in such

218

Bhardwaj, T. and Jha, S.

Microﬂuidic Platform for Aptamer based Fluorimetric Analysis of Analytes.

DOI: 10.5220/0006645002180223

In Proceedings of the 11th International Joint Conference on Biomedical Engineering Systems and Technologies (BIOSTEC 2018) - Volume 1: BIODEVICES, pages 218-223

ISBN: 978-989-758-277-6

sandwich protocol required sophisticated

instrumentation including a microscope (Tennico,

2010; Fenzyl, 2016). Hence, in our work, we

developed a simple setup for fluorescence detection

using PMMA-PMMA microfluidic chip and

aptamers as probe.

The choice of polymethyl methacrylate (PMMA)

slides as a substrate was due to its sturdiness and

possibility for immobilization of aptamer as a probe.

PMMA is also a choice in fabrication of

microfluidics chips because of its low cost and

optical clarity (Tennico, 2010). Various techniques

are available for fabrication of microfluidic chips

like lithography, micromachining, laser ablation,

thermal embossing etc. While, in this work, we used

simple techniques like laser ablation for cutting of

and engraving of channels on PMMA, and thermal

and UV bonding for sticking the two layers.

Apart from developing a PMMA microchip, we

also developed a simple setup for fluorescence

detection. Majorly, the strategy employed for

detection of various analytes using aptamers

involves simple hybridization with dye labeled

complementary nucleic acid or sandwich assay.

Here, we have used both the strategies to check our

fluorescence detection setup. Aptamers were

immobilized using hexamethylene diamine (HMDA)

and glutaraldehyde for both the strategies. For

hybridization, we used an aminated sequence of

aptamer whose complementary 6-carboxyfluorescein

(FAM) labeled strand was made to interact. While in

other strategy, thrombin was used as a model analyte

to validate sandwich assay using our setup. The

setup for fluorescence detection is shown in Figure

1. This setup enabled low cost fluorescence

detection without use of bulky microscopes. In

addition, this can be miniaturized further via using a

different detector and LabVIEW software.

2 MATERIALS AND METHODS

2.1 Materials

All chemicals were of analytical grade and

purchased from Sigma or Merck. Aptamer sequences

were purchased from Integrated DNA Technologies

(IDT). Sequences for first and second strategy were :

5’-/5AmMC6/GCC AAA TTG TTT GAC GAG A-

3’, 5’-/56-FAM/ TCT CGT CAA ACA ATT TGG

C-3’ and 5’-/5AmMC6/ TTT TTG GTT GGT GTG

GTT GG-3’, 5’-/56-FAM/TTT TTT TTT TTT TTT

AGT CCG TGG TAG GGC AGG TGG GGG TGA

CT-3’. Cyan LED 490nm, PMMA sheets and optical

narrow bandpass interference filter of 530 nm were

purchased from eBay India Online Store and Optics

& Allied Engg. Pvt. Ltd. respectively.

2.2 PMMA-PMMA Chip Fabrication

PMMA sheets of thickness 1.5 and 5 mm were cut

into substrates of 60 x 20 mm using CO

laser

cutting machine. Same technique was used to

engrave channel of depth 130µm, puncture for inlet

tubing, sensing area and outlet tubing, and detection

area. The detection area was made in such a way that

it will be at 90º angle with LED source for

fluorescence detection. Further, substrates were

ultrasonicated in Isopropyl alcohol (IPA) for 20

minutes and dried using nitrogen purging.

For bonding of both layer of substrates, UV and

Figure 1: Fluorescence detection setup used with microfluidic chip.

Microﬂuidic Platform for Aptamer based Fluorimetric Analysis of Analytes

219

solvent assisted thermal bonding were used. First,

ethanol was poured on both substrates, aligned over

each other avoiding any air bubble, clipped properly

and kept for UV bonding for 20 minutes. Then, after

removing clips, chips were kept on hot plate for 2

hours under 1 Kg weight at 120ºC. Further, weights

were removed when substrate cooled to room

temperature. Next, they were soaked in IPA for 10

minutes and rinsed with double distilled water.

Design of PMMA-PMMA microfluidic chip is

shown in Figure 2. Here, detection area was an

aperture in PMMA substrate at 90º to sensing area

where optical fiber was inserted for fluorescence

detection. Sensing area is the area where aptamers

were immobilized for interaction with analyte. Inlet

and outlet in the microchip were made for insertion

of inlet and waste tubing.

Figure 2: Design of PMMA-PMMA microfluidic chip.

2.3 Sensing Area Modification and

Aptamer Immobilization

Sensing area on PMMA was treated with a solution

of 10% HMDA in 100 mM borate buffer of pH 11.5

for 2 hours. Following this, the surface of sensing

area was washed 3-4 times with distilled water.

Afterwards, surface was activated with 2.5%

glutaraldehyde in 0.1 M phosphate buffer pH 7 for 2

hours at room temperature followed by washing with

0.1 M phosphate buffer. Then, aminated aptamer

sequences were drop casted on sensing area for

immobilization and kept for 1 hour at room

temperature. Next, channels were washed with 1×

Tris EDTA (TE) buffer using syringe pump with

flow rate of 5 µl/min. In our two different strategies,

different sequences were immobilized using same

protocol. Immobilization step was confirmed by

immobilizing an aptamer 5’-/5AmMC6/GCC AAA

TTG TTT GAC GAG A-3’ and passing FAM

labeled complementary strand 5’-/56-FAM/ TCT

CGT CAA ACA ATT TGG C-3’. Fluorescence

intensity from sensing zone was checked before and

after passing complementary strand into the

microchannel.

2.4 Instrumentation

The instrumentation involved in our setup had a

Cyan LED of 490nm as excitation source. LED was

placed just above sensing area which was at 90º to

detection area. Optical fiber was inserted into

detection area of PMMA-PMMA chip followed by

an optical narrow bandpass interference filter of 530

nm. This optical fiber passed fluorescence signal

picked from the sensing zone of the microchip. Next,

the signal from filter was further collected at the

photodetector (Ocean Optics HR2000+) and changes

were read via commercial Spectrasuite software

from Ocean optics.

2.5 Sensing Procedure

In our first strategy, immobilized aptamer 5’-

/5AmMC6/GCC AAA TTG TTT GAC GAG A-3’

was made to interact with different concentrations of

complementary FAM labeled DNA 5’-/56-FAM/

TCT CGT CAA ACA ATT TGG C-3’. Concentra-

Figure 3: Increase in fluorescence after passing FAM labeled complementary ssDNA.

BIODEVICES 2018 - 11th International Conference on Biomedical Electronics and Devices

220

tion from 0.6-1000ng of complementary DNA was

passed through channel for 45 minutes and then

channels were washed with 1×TE buffer for 15

minutes. Both steps were performed at 5µl/min.

Fluorescence was noticed before and after passing

complementary DNA.

For confirmation of sandwich assay protocol,

immobilized primary aptamer 5’-/5AmMC6/ TTT

TTG GTT GGT GTG GTT GG-3’ was made to

interact with model analyte thrombin. Different

concentrations of thrombin, from 6-600 ng were

passed through channel for 45 minutes and then

channels were washed using 1×TE buffer for 15

minutes. Both steps were performed at 5 µl/min.

Afterwards, FAM labeled secondary aptamers 5’-

/56-FAM/TTT TTT TTT TTT TTT AGT CCG TGG

TAG GGC AGG TGG GGG TGA CT-3’ were

passed over the detection zone. Fluorescence

intensities from detection zone were recorded before

and after passing secondary aptamers.

3 RESULTS AND DISCUSSION

3.1 Confirmation of Sensing Protocol

We checked immobilization of aptamer by passing

its FAM labeled complementary strand in the

microchannel post immobilization of aptamer in the

sensing area. We found an increase in fluorescence

intensity post hybridization as shown in Figure 3.

This increase shows that hybridization occurred on

binding of FAM labeled complementary DNA strand

which increased number of FAM molecules in

sensing area. Hence, it proved successful

immobilization of aptamer.

Further, we tested our first strategy of

fluorescence detection using simple hybridization of

FAM labeled complementary strand. Increase in

fluorescence was seen with increase in concentration

of complementary ssDNA as shown in Figure 4. A

linear calibration curve was also obtained for the

same, as shown in Figure 5. Both figures show

proportionate increase in fluorescence with increase

in concentration of complementary DNA strand.

When complementary DNA strand was passed

through channel of microfluidic chip, it hybridized

with immobilized aptamer. According to the

arrangement in our setup, when FAM labeled

complementary DNA was targeted with light of cyan

LED source, dye molecules got excited and showed

fluorescence which was detected at 90º angle to

detection area using attached fiber optic probe and

coupled CCD array spectrometer. Therefore, it

proved direct relationship between concentration of

analyte and fluorescence. According to figure 5, the

linear range of detection was between 6 to 1000 ng

of ssDNA with 6 ng as the practical LOD for

measurement. Hence, this setup could be used for

detection of nucleic acid sequences using simple

hybridization.

3.2 Sandwich Assay Protocol

As our second detection strategy, we tested

fluorescence detection using thrombin protein to

validate sandwich assay using primary and FAM

labeled secondary aptamers. Again, increase in

fluorescence was observed proportionate to the

concentration of analyte thrombin as seen in Figure

6. When thrombin was passed through microchannel

in microfluidic chip, it interacted with its

immobilized primary aptamer. Then, FAM labeled

secondary aptamer for thrombin was passed through

channel. This secondary aptamer attached wherever

thrombin analyte was bound to primary aptamer and

hence, any increase in fluorescence was directly

related to thrombin concentration, as secondary

aptamer would not bind in absence of thrombin in

sensing area. As per figure 6, the linear range of

detection was found as 125-600 ng with 125ng as the

practical LOD for measurement. Therefore, the

Figure 4: Increase in fluorescence proportionate with concentration of complementary ssDNA.

Microﬂuidic Platform for Aptamer based Fluorimetric Analysis of Analytes

221

Figure 5: Calibration curve for complementary ssDNA.

Figure 6: Increase in fluorescence proportionate to increasing concentration of thrombin.

protocol was suitable for detection of diseases such

as some types of cancer, microbial diseases,

myocardial infarction etc. that gives rise to altered

concentration of biomarkers such as protein in blood

(Viscidi, 1987).

4 CONCLUSION

We have reported a simple and low cost setup for

fluorescence detection using aptamers as probe

without using any bulky devices like microscope.

We fabricated a PMMA-PMMA microfluidic chip

using simple and easily available laboratory

techniques and combined the chip with a simple

fluorescence detection setup. In this new approach,

we used two different strategies to use aptamers as

probe. In first strategy, diseases which can be

identified by nucleic acid hybridization could be

analyzed. We proved this using known sequence of

aptamer and its FAM complementary strand. Here,

we found that whole setup showed reliable linear

range of detection. In other strategy, diseases that

can be identified using analysis of any proteinic

biomarker could be detected using our sandwich

technique. In this, we used thrombin as model target

to validate our setup. We found that the minimum

concentration of thrombin which could be detected

out using our setup was impressive. Both the

strategies proved successful with our setup. In

future, this setup size could further miniaturized by

using a small CCD array, microcontroller and

LabVIEW software.

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