10.1.1.136.3467

8/12/2019 10.1.1.136.3467

1/9

1

HIGHLY SENSITIVE,

RAPID, RELIABLE, AND AUTOMATICCARDIOVASCULAR DISEASE DIAGNOSIS WITH

NANOPARTICLE FLUORESCENCE ENHANCERAND MEMS

Bin Hong, a Junhai Kai, b Yongjie Ren, a Jungyoup Han, b Zhiwei Zou, b Chong H. Ahn, b and Kyung A. Kang a

1. INTRODUCTION

Cardiovascular diseases, especially acute myocardial infarction (MI; heart attack),have been the leading threat to human life. 1 Thus, in the emergency situation, a rapid andaccurate diagnosis of cardiovascular diseases is important to save lives. It can be realized

by rapidly and sensitively measuring cardiac markers that are released from injuredcardiac muscles. Creatine Kinase-MB (CK-MB), myoglobin (MG), and cardiac troponinI (cTnI) are important markers for early diagnosis of heart attack. 2 B-type natriuretic

peptide (BNP) and C-reactive protein (CRP) are crucial markers for diagnosis and prognosis of congestive heart failure (CHF) and acute coronary syndromes (ACS),respectively. 3-4 Our effort is focused on developing highly sensitive, accurate, andreliable instrument using nanoparticle reagents and Microelectromechanical Systems(MEMs).

The main challenge in developing biosensor is the low concentrations of cardiac

markers in plasma (only a few tens pico-moles) at an early stage of cardiovasculardiseases. 5 Since our sensing is interrogated by fluorescence, fluorescence enhancementcan, therefore, improve the sensitivity. Nanogold particles (NGPs) hold strong plasmon

polariton field on their surface, which can couple the lone-pair electrons (normally usedfor self-quenching) of the fluorophore for fluorescence enhancement. 6-7 Some

a Bin Hong, Yongjie Ren, and Kyung A. Kang, Department of Chemical Engineering, University of Louisville,Louisville, KY 40292.

b Junhai Kai, Jungyoup Han, Zhiwei Zou, and Chong H. Ahn, Department of Electricaland Computer Engineering and Computer Science, University of Cincinnati, Cincinnati, OH 45221.

8/12/2019 10.1.1.136.3467

2/9

B. HONG ET AL . 2

biocompatible solvents were also found to enhance fluorescence, by shifting thefluorophore excitation/emission wavelength and increasing the amount of trans carbondouble bonds. 6-7 To maximize the enhancement effect, NGPs and the solvent werecombined, forming nanogold particle reagents (NGPRs). According to our previousresults, the mixture of 5 nm sized NGPs coated with 2 nm thick self-assembledmonolayer (5nmNGP-SAM2nm) and 1-butanol has shown to be an excellent enhancer. 7

MEMs improves biosensors by ultra-small sample usage, high sensing consistency,high compactness, as well as mass production and low energy consumption. Thus, for areliable and fully automated sensing performance with a minimal system size, MEMswas integrated to the sensing system. Our research group has previously developed a

prototype, semi-automatic, four-marker sensing system. 8 Using this system simultaneousfour-cardiac marker quantification was completed within 10 minutes. The averagesignal-to-noise (S/N) was also as high as 20, which was excellent.

In this paper, based on our previous system, a more sensitive and accuratesimultaneous four-cardiac marker sensing system with the application of NGPR andMEMs is reported.

2. MATERIALS, INSTRUMENTS, AND METHODS

2.1. NGP, solvent and NGPR related study

5 nm nanogold particles coated with tannic acid (Ted Pella, Redding, CA) and 16-mercaptohexadecanoic acid (MHA; Sigma/Aldrich, St. Louis, MO) were used to

synthesize 5nmNGP-SAM2nm by self-assembling MHA on NGP. 6 For the NGPR with butanol basis, 5nmNGP-SAM2nm was then dispersed in 1-butanol (Sigma/Aldrich).

2.2. Cardiac marker sensors and assay protocol

Human BNP was purchased from Bachem (Torrance, CA) and monoclonal IgGsagainst human BNP, from Strategic Biosolutions (Newark, DE). cTnI, MG, CRP fromhuman heart and their respective monoclonal antibodies were obtained from FitzgeraldIndustries (Concord, MA). The fluorophore, Alexa Fluor 647 (AF647; max.excitation/emission wavelengths, 649/666 nm) was from Invitrogen (Carlsbad, CA).Four cardiac marker biosensors were constructed following the protocol established byTang et al .5 The fluorometer with four sensing channels (Analyte 2000 TM) was purchasedfrom Research International (Monroe, WA). The main principle of our sensing system is:

The first monoclonal antibodies (1o

Mab) against the respective marker are covalentlyimmobilized on the sensor surface. During the assay, the sample is injected to thesensing chamber and the target marker binds specifically to the 1 oMab. All otherunbound molecules are washed away from the sensing chamber. Next, the secondmonoclonal antibody tagged with AF647 (AF647-2 oMab) is applied to the sensor and

binds to the 1 oMab-marker complex, forming sandwich complex. When the boundAF647s are excited by the light source, the fluorescence is emitted from the sandwichcomplex and the intensity is correlated with the marker concentration in samples.Sensing with NGPR follows the protocol established by Hong and Kang: 6 NGPR isapplied before the sample incubation. During the incubation of the NGPR, the

8/12/2019 10.1.1.136.3467

3/9

MULTI-CARDIAC MARKER BIOSENSOR 3

Figure 1. Schematic diagram of hot embossing using PDMS mold and thermal bonding. The plastic substrateis heated to a temperature above the glass transition temperature (Tg) of the polymer material and a masterPDMS mold is pressed against the molten side of the substrate. The substrate cools as the force is applied andthe pattern on the PDMS mold is transferred to the plastic substrate.

fluorescence signal is measured for the base line. NGPR is also applied after theincubation of AF647-2 oMab and sensor washing. The fluorescence is measured again forthe sample. Then, the difference of these two measurements is correlated to the markerconcentration in sample.

2.3. Microfluidic sensing system utilizing MEMs

Labview TM (version 7.1) and data acquisition (DAQ) card (USB-6008, 8 inputs, 12 bits, 10 ks/s, multifunctional I/O) from National Instruments (Austin, TX) were used tocontrol the entire sensing system. Electronically controllable micro-solenoid pump (12 v,50 L per stroke, 2 W) and micro-solenoid valves (12 v, 280 mW) from The Lee Co.(Westbrook, CT) execute microfluidics. Drive circuit with power plug, switch and LEDwere customized by our research group. The plastic microchannel network andserpentine sensing module were microfabricated as follows: 9 Thick photoresist (SU-8) on

nickel disc (diameter, 3 inch) was patterned by UV-LIGA process; Polydimethylsiloxane(PDMS) was cast on nickel disc for PDMS mold with desired pattern (Fig. 1); Hotembossing between the plastic wafer and PDMS mold was performed to transfer the

pattern on plastic wafer; Plastic wafers were sliced; Thermal bonding of multiple plasticwafers was conducted for microchannels. To generate micro-turbulence, bumps or

baffles were added on the upper and bottom sides of a microchannel with a square cross-section for a serpentine sensing chamber.

3. RESULTS AND DISCUSSION

Our fluorophore mediated, fiber-optic immuno-sensor is a highly sensitive detectiontool. As an example, from our comparative study for BNP quantification, it has a

sensitivity 100 times higher than Enzyme-Linked Immunosorbent Assay (ELISA) (datanot shown). Because of its sensitivity and accuracy, it may be used for various humandisease diagnosis/ prognosis. 5,8,10 However, for rapid and simultaneous multi-cardiacmarker quantification, additional sensitivity improvement is needed, especially for BNPand cTnI due to their extremely low concentrations in plasma at an early disease stage.

3.1. Cardiac marker sensing using NGPR

As previously stated, 5nmNGP-SAM2nm in 1-butanol was an excellent NGPR. Itsenhancement effect was, therefore, tested in a 3-cm BNP sensor in a smooth capillary

8/12/2019 10.1.1.136.3467

4/9

B. HONG ET AL . 4

(a ) (b )

Figure 2. Sensing performances of (a) BNP sensor at the BNP sensing range and (b) four cardiac markersensors in the microfluidic sensing system without using NGPR and with NGPR. [Experimental conditions: for(a), 3-cm BNP sensor, 3/4 minutes for BNP and AF647-2Mab incubation, respectively, flow velocity at 1.2cm/sec, pump frequency at 0.6 Hz, NGPR, 5nmNGP-SAM2nm in 1-butanol, capillary microchannel, automaticsensing; For (b), size of four sensors, 3 cm, plasma sample with 0.1 ng/mL BNP; 0.7 ng/mL cTnI; 70 ng/mLMG; and 700 ng/mL CRP, AF647-2Mab mixture, 20 g/ml in PBS buffer for all markers, serpentine sensingmodule, other conditions were same as (a).]

microchannel. Figure 2a shows the sensing performance of BNP sensor with and withoutthe NGPR. With the NGPR, the sensors sensitivity was found to be 410% more thanthat without NGPR in average. This NGPR was also tested with four sensors encased ina four-microchannel serpentine sensing module (Fig. 2b). To examine the enhancementeffect of the NGPR for four different sensors simultaneously, the mixture of four cardiacmarkers in plasma was used as a sample. The concentration of each marker was selectedto be at its lower limit in its sensing range, as this condition is most difficult to measureand the signals are most needed to be enhanced. Results showed that NGPR is able ofincreasing the sensitivities of BNP, cTnI, MG, and CRP sensors by 60, 50, 180, and230%, respectively, indicating that signals from the sensors for the markers with higherconcentration ranges were enhanced more than those for lower concentration markers. Itis probably because relatively larger amount of fluorophore tagged protein complexes

bound on sensor surface due to high concentration of the marker in sample provide moreopportunity of interaction with NGPR for fluorescence enhancement. In our studies,

NGPRs have shown their potential as nano-fluorescence enhancers in various fluorophoremediated, fiber-optic, biomarker sensors whether the marker is protein (e.g., MG) or

peptide (e.g., BNP). 5,8

3.2. Cardiac marker sensing using MEMs

For automatic sample and fluid delivery, circulation, and washing in our system, acomputer assisted flow control unit was developed (Fig. 3ab). In the unit, there are amicro-pump, seven micro-valves, and a serpentine sensing module which are connected

by the microchannel network forming an open (washing)/ close (sample and fluiddelivery, circulation) flow pattern. A computer code written by Labview TM software wasthen added to electronically control these electronic parts for an easy operation.Therefore, a MEMs was constructed for simultaneous four-cardiac marker quantification

0

100

200

300

400

500

600

700

0 50 100 150 200 250 300

BNP concentration (pM)

S i g n a l i n t e n s i t y

( p A )

Without NGPR

With NGPR

0

100

200

300

400

500

600

700

800

900

BNP cTnI MG CRP

S i g n a

l i n t e n s

i t y

( p A )

Without NGPR

With NGPR

8/12/2019 10.1.1.136.3467

5/9


with our sensing protocols (Fig. 3c). This system is beneficial not only for a user-friendly diagnostic tool, but also improving the reliability of the system since itminimizes human errors. Automated assays showed similar signal intensities to those ofthe manual operation (less than 5% discrepancy) but doubled average S/N ratio from 18to 35 (data not shown). In addition, by using microchannel network, the sample volumerequired for each assay was decreased from 1 mL to 300 L.

Effective analyte mass transport from bulk media to sensor surface is important formass-transfer-limited, fiber-optic, immuno-biosensing. Convective application of liquidsamples/ reagents to the sensor surface was proven to improve the sensitivity of this typeof sensors. 10 However, the laminar flow in the smooth surfaced capillary channels still

limits the analyte transport.5

The microchannel with the inner surface that can createlocal turbulence may enhance the analyte transport to the surface. 8,11 For this purpose, aseries of bumps (radius, 600 m; height, 400 m; spacing between adjacent bumps, 1200 m) were microfabricated on the inner surface of the microchannel (i.e., serpentinemicrochannel) (Fig. 4abc). Out of several bump shapes, half-circular bump was found to

be very effective. 8 By applying this serpentine microchannel, the sensing performance of3-cm BNP and MG sensors were studied and the results were compared with those usingsmooth capillary microchannel. BNP and MG were selected because one has a lowconcentration range (26~260 pM) and the other, a high concentration range (4~40 nM).For BNP sensing (Fig. 4d), serpentine microchannel presented approximately 30~90%

Figure 3. BioMEMs: (a) Schematicdiagram and (b) the top view of themicrofluidic sensing unit: plastic platewith imbedded microchannel network,micro-pump, micro-valves, serpentinesensing module; (c) BioMEMscomponents: laptop computer withLabVIEW control panel, microfluidicsensing unit with serpentine sensingmodule, Analyte 2000 (fluorometer)with four sensing channels.

2-way microvalves

3-waymicrovalve

Micropump

Microchannelnetwork

Plastic plateWaste outlet(a )

Optical sensors

Serpentine sensing module

2-way microvalves

Optical sensorsSerpentinesensing module

(b )

LabVIEWcontrol panel

Microfluidicsensing unit

Fluorometer (c)

8/12/2019 10.1.1.136.3467

6/9

B. HONG ET AL . 6

higher signals than capillary microchannel. While for MG sensor, a slight signal increase(0~6%) was exhibited (Fig. 4e). It may indicate that serpentine structure showed better

performance for BNP since BNP has low sensing range and, therefore, the mass transportwas more limited than MG.

In order for our sensing system to be used for rapid disease diagnosis, a shorter assaytime is desired. Initially, the incubation times for the sample and the AF647-2 oMab withconvection were 3 and 4 min, respectively, which lead to a total assay time of 10 minincluding washing and regeneration steps. To increase the sensitivity and minimize theassay time, NGPR and MEMs were incorporated into the sensing system. The sensing

performance of four sensors was, therefore, studied simultaneously in the four-channel plastic serpentine sensing module (Fig. 4e) at various incubation times for the sample andthe AF647-2 oMab mixture for four markers. For the effect of the sample incubation timeon the sensing performance, plasma sample with four markers at their lowerconcentration limits was incubated for 1, 2 or 3 min. For this test, the incubation time for

(b )

Serpentine bumps

4 -channelsensingmodule

Microchannels (c)

Figure 4. Schematic diagram (a) and the top view (b) of aserpentine microchannel [Structure: a series of half-circular bumps at 600 m radius, 400 m height and 1200 m spacing between bumps and 1.4 1.4 mm squarecross-section]; and (c) a four-channel serpentine sensingmodule; and the sensing performance of (d) BNP and (e)MG sensors using capillary microchannel ( ) andserpentine microchannel ( ). [Experimental conditions: 3-cm BNP or MG sensor, 3/4 minutes for sample andAF647-2Mab incubation, respectively, flow velocity at1.2 cm/sec, pump frequency at 0.6 Hz, NGPR, 5nmNGP-SAM2nm in 1-butanol , automatic sensing, single channelserpentine sensing module with the structure same as (a).]

600 m

1200 m

1.4 mm

1.4 mm

400 m

(a )

0

100

200

300

400

500

600

700

800

900

0 50 100 150 200 250 300

BNP concentration (pM)


( p A )

Capillary microchannel

Serpentine microchannel

(d )

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

0 10 20 30 40 50

Myoglobin concentration (nM)


( p A )

Capillary microchannel

Serpentine microchannel

(e)

8/12/2019 10.1.1.136.3467

7/9


Figure 5. The effects of (a) sample reaction time and (b) AF647-2Mab reaction time on the sensing performance of four sensors. [Experimental conditions: four cardiac markers with concentrations at the lowerlimit in their sensing ranges, 0.1 ng/ml BNP, 0.7 ng/ml cTnI, 70 ng/ml MG, 700 ng/ml CRP in plasma; sensorsize, 3 cm; incubation time of AF647-2Mab mixture was fixed as 4 min for the effect of sample reaction time,while sample mixture incubation time was fixed as 3 min for the effect of AF647-2Mab reaction time; flowvelocity, 1.2 cm/sec; NGPR, 5nmNGP-SAM2nm in 1-butanol; serpentine sensing module; automatic sensing.]

the AF647-2 oMab mixture remained constant at 4 min (Fig. 5a). Due to the similarsensing range, results of BNP and cTnI sensors were shown in one figure, while MG andCRP in another figure. Sensing of cTnI (Fig. 5a1, ) and MG (Fig. 5a2, ) presentedsimilar exponential increase of signals as the sample reaction time increased. Thereaction between the analyte and its corresponding 1Mab was minimal before 2 minwhile increased significantly after 2 min. While for BNP (Fig. 5a1, ) and CRP (Fig.5a2, ) sensors, signals increased nearly linearly with the increase of the sampleincubation time, indicating a faster reaction for CRP and BNP. Although the signal mayincrease after 3 min for all sensors, the signal intensities from four sensors were much

higher than those without using NGPR and serpentine sensing module. When thereaction time was 2 min with NGPR and serpentine sensing module, the signals were1.3~6.4 times of the signals without using NGPR or serpentine sensing module.Therefore, 2 min reaction time was enough for the sample incubation for accuratequantification of four markers simultaneously. Similarly, with a constant sampleincubation time of 3 min, the effect of the AF647-2Mab incubation time (1, 2, 3, or 4min) on the sensing performance was studied. All four sensors showed a similar signal

profile with the increase of the AF647-2Mab incubation time (Fig. 5b). At 3 min, thesignal increase was reduced significantly. 2 min showed signal intensities 110~450% ofthose by 4 min reaction time without NGPR or serpentine sensing module. Therefore, the

0

100

200

300

400

500

600

700

800

900

0 1 2 3 4

Sample mixture incubation time (minute)


( p A )

MG

CRP

0

100

200

300

400

500

600

700

800

900

0 1 2 3 4 5

AF647-2Mab mixture incubation time (minute)


( p A )

MG

CRP

(a2 )

(b2 )

0

10

20

30

40

50

60

70

0 1 2 3 4

Sample mixture incubation time (minute)


( p A )

BNP

cTnI

0

10

20

30

40

50

60

70

0 1 2 3 4 5

AF647-2Mab mixture incubation time (minute)


( p A )

BNP

cTnI

(a1 )

(b1 )

8/12/2019 10.1.1.136.3467

8/9

B. HONG ET AL . 8

total assay time was shortened by 3 min from the previous protocol of without using NGPR and serpentine sensing module.

4. CONCLUSIONS

An automated, fluorophore mediated, fiber-optic, multi-analyte, immuno-sensingMEMs system was developed to quantify four important cardiac markers simultaneouslyin blood plasma. To improve the system sensitivity, fluorescence enhancing NGPR wasapplied and the sensitivities of BNP, cTnI, MG and CRP sensors were increased byapproximately 60%, 50%, 180% and 230%, respectively. Serpentine structure wasmicrofabricated on the surface of the sensing microchamber and the structure could alsoimprove the sensitivity. MEMs was incorporated to the system for a reliable detectionand user-friendly operation. As a result, the consistency of the system was doubled; theassay time was shortened to be 7 min and; the sample volume decreased to 300 L.

Our multi-analyte Biosensing MEMs is potential for quantifying any group of humandisease representing biomarkers rapidly, simply, and cost-effectively.

ACKNOWLEDGEMENT

Authors acknowledge the financial supports from Kentucky Science and EngineeringFoundation (KSEF-148-502-03-55) for fluorescence enhancement studies and NationalScience Foundation (BES-0330075) for cardiac marker biosensing. Sigma Xi honor

society is acknowledged for Bin Hongs Grants-in-Aid of Research award for NGPsrelated studies and the Institute for Molecular Diversity and Drug Design (IMD 3) at theUniversity of Louisville for Bin Hongs Graduate Fellowship.

REFERENCES

1. American Heart Association. Heart Disease and Stroke Statistics, Update, 10-12 (2005).2. F. S. Apple, R. H. Christenson, R. Valdes, A. J. Andriak, K. Mascotti, and A. H.B. Wu, Simultaneous rapid

measurement of whole blood myoglobin, creatine kinase MB, and cardiac troponin I by the triage cardiac panel for detection of myocardial infarction, Clin. Chem. 45 (2), 199-205 (1999).

3. A. S. Maisel, P. Krishnaswamy, H. C. Herrmann, and P. A. McCullough, Rapid measurement of B-typenatriuretic peptide in the emergency diagnosis of heart failure, New Engl. J. Med. 347 , 161-167 (2002).

4. M. S. Sabatine , D. A. Morrow, C. P. Cannon, and E. Braunwald, Multimarker approach to risk stratificationin non-ST elevation acute coronary syndromes: Simultaneous assessment of troponin I, c-reactive protein,

and b-type natriuretic peptide, Circ. 105 , 1760-1763 (2002).5. L. Tang, Y. J. Ren, B. Hong, and K. A. Kang, A fluorophore-mediated, fiber-optic, multi-analyte, immuno-

sensing system for rapid diagnosis and prognosis of cardiovascular diseases, J. Biomed. Optics 11, 021011 (2006).6. B. Hong and K. A. Kang, Biocompatible, nanogold-particle fluorescence enhancer for fluorophore mediated,

optical immunosensor, Biosens. Bioelectron. 21 (7), 1333-1338 (2006).7. K. A. Kang and B. Hong, Biocompatible nano-metal particle fluorescence enhancers, Crit. Rev. Eukar. Gene

Expres. 16 (1), 45-60 (2006).8. L. Tang, 2005. Multi-analyte, fiber-optic immuno-biosensing system for rapid disease diagnosis: model

systems for anticoagulants and cardiac markers. Dissertation. Chemical Engineering, University ofLouisville, Louisville, KY

9. Y. Sohn, J. H. Kai, C. H. Ahn, Protein array patterning on Cyclic Olefin Copolymer (COC) for disposable protein chip, Sensor Lett. 2, 171-174 (2005).

8/12/2019 10.1.1.136.3467

9/9


10. L. Tang, H. J. Kwon, and K. A. Kang, Theoretical and experimental analysis of analyte transport in a fiberoptic, protein C immuno-biosensor, Biotech. Bioeng. 88 , 869-879 (2004).

11. J. S. Rossier, G. Gokulrangan, H. H. Girault, S. Svojanovsky, G. S. Wilson, Characterization of proteinadsorption and immunosorption kinetics in photoablated polymer microchannels, Langmuir 16 , 8489-8494 (2000).

10.1.1.136.3467

Documents