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titatively with high sensitivityi.e., creatinine concentration as

low as 0.5 g /mL. Preparation of these SERS active sub-strates are inexpensive and easy to prepare.1113 The creatininelevels of urine samples from diabetic patients and healthypersons were measured quantitatively using metalized nano-

structured parylene substrates. As a result, we showed thesuitability of using our SERS substrates in urine analysis.

2 Results and DiscussionWe recently demonstrated that nanostructured polychloro-p-xylylene PPX-Cl, also known as parylene films can be fab-ricated by the oblique angle polymerization OAPmethod.1113 The nanostructured PPX films are deposited on asubstrate from a directional vapor source. Vapor depositionand polymerization at an oblique angle relative to the sub-strate surface permits the fabrication of films possessing nano-structured morphology. The nanostructured PPX film com-prises free-standing, slanted, parallel columns containingnanowires. Subsequently, we deposited a thin layer i.e.,

60 nmof Ag metal film onto the nanostructured PPX tem-plate. Figure 1a shows the metalized nanostructured PPXfilm coated on a glass slide. Figure1bshows the schematics

of nanostructured PPX film metallization. Scanning electronmicroscopeSEM images show the nanostructured morphol-

ogy of the metalized PPX film at two different magnifications

Figs. 1c and1d. Ag nanoparticles 60 nm cover thenanostructured PPX films uniformly. The SERS substrates

have high uniformity5% signal variation in a1 mm2 areaand sample-to-sample reproducibility.14 The enhancement fac-

tor EF was calculated as 105 for the Ag metalizedsubstrates.14,15

Raman spectra of pure creatinine, artificial urine, and urinefrom a diabetic patient were measured on the nanostructured

PPX-Cl substrate. Significant peaks at 700, 840, 900, and

1420 cm1 were observed in the Raman spectra of creatinineFig. 2a. However, in urine measurements, we found that

840- and 900-cm1 peaks are stronger than the 700-cm1

peak.A significant advantage of our nanostructured substrate

over traditional metal colloids is the enhanced stability of Ra-man signals when detecting samples with different ionic

strengths. The variation of the ionic strength of the analytecan affect the aggregation process of metal colloids. It has

been reported that the reproducibility of Raman signal dropsdrastically if the salt content of the urine varies.8 In contrast,our nanostructured SERS substrates show superior stability

due to the immobilized metal nanoparticles on the surface ofthe SERS surface. Figure2bshows robustness of SERS sig-

nal with various potassium chlorideKClconcentrations. The

urea peak at 1000 cm1 shows an intensity variation of lessthan 5%. Each spectrum is shifted for clarity. A concentrationprofile for varying creatinine concentration in artificial urine

is measured.The reproducibility of our SERS substrate is demonstrated

using 1 to 3 l of urine sample from a diabetic patient. 12spots were chosen randomly on the surface of the nanostruc-tured substrate to collect Raman spectra Figure 3a. From

spot to spot, the urea signature peak at 1000 cm1 symmetri-cal C-N stretch shows an intensity variation of less than 5%.This major peak can be used as an internal reference for urine

c" d"

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Fig. 1 a Surface-enhanced Raman substrate and b schematic ofSERS substrate preparation are shown. The surface area of the SERS-active region is approximately 1 cm2. SEM image of the Ag-PPX-ClSERS substrate at clow magnification 5500 and dhigh magni-fication20,000. Scale bar for SEM images is 1 m.

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Fig. 2 aSERS spectra of: a creatinine solution 0.5 mM top, artificial urine solution composed of urea, creatinine, and uric acid middle, anda urine sample bottom. Artificial urine concentrations are 1639, 104, and 34 mg/dl, respectively, which is approximately the same level ofhealthy human urine.18 All SERS measurements are colleted at 10-s acquisition time with four scans. bSERS spectra taken with different amountsof KCl added to a urine sample. Concentrations of KCl from top to bottom are 0 M, 0.5 mM, 5 mM, 50 mM, and 0.5 M, respectively 10-sacquisition time and four scans.

Wang et al.: Quantitative analysis of creatinine in urine by metalized nanostructured parylene

Journal of Biomedical Optics March/April 2010 Vol. 152027004-2

Downloaded from SPIE Digital Library on 25 Mar 2010 to 130.203.253.62. Terms of Use: http://spiedl.org/terms

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analysis, considering the fact that urea is the dominant organiccomponent of human urine, and the excretion rate of urea inurine is relatively stable. Figure 3b shows SERS spectra of

creatinine at 0.5-, 5.1-, and 10.2-g /mL concentrations, re-spectively. SERS intensity at 840 and 900 cm1 are measuredfrom artificial urine samples. These results also show that

creatinine concentrations as low as 0.5 g /mL can be de-tected easily by SERS method using our nanostructured sub-strate. Mass spectroscopy technique, commonly used in clini-cal chemistry, can detect creatinine concentrations of

0.5 g /mL, with an uncertainty of approximately 3%.16 Oursubstrate provides a similar sensitivity and reproducibility;hence it may be used as a quantitative technique in clinicalmeasurements in the future.

Table 1 provides details of Raman measurement param-eters and creatinine concentrations measured by enzymaticassays. The creatinine concentrations of clinical urine samplesare measured by Diazymes enzymatic creatinine reagent kit.This assay is known to have no interference from ascorbicacid, bilirubin, hemoglobin, or triglycerol. In this assay, crea-tinine is converted into creatine, and then transferred into sar-cosine. The sarcosine is oxidized to hydrogen peroxide, whichis measured by a Trinder reaction.

Figure 4a shows the quantitative measurement of creati-nine from diabetic and healthy patients. Two peaks at 840 and900 cm1 are chosen in the SERS spectra for the quantitativeanalysis of creatinine. Creatinine has two major peaks at 840and 900 cm1, but there is spectral overlap from other com-ponents of urine in the same region e.g., urea and creatinine

at 840 and900 cm1. Therefore, the SERS data are analyzedby an ordinary least-squares method to generate linear predic-tive models of creatinine concentration of urine samples.There is a linear relationship between creatinine concentra-tions and SERS peak areas at 840 and 900 cm1. Percentageof varianceR2 equals 0.907 and 0.967, for 840 and900 cm1

data, respectively. The linear model is also analyzed with re-

sidual plots,17 and the p-values were calculated by theAnderson-Darling test. We conclude that the residuals are nor-

mally distributed, since the p-values 0.514 and 0.245 forSERS data at 840 and 900 cm1, respectively are greater

than 0.05 level of significance. Figure4b shows the crea-tinine concentrations measured by SERS and the enzymaticmethod. The SERS data are in good agreement with the en-zymatic data. The correlation coefficients are 0.907 based on

the SERS peak at 840 cm1 and 0.968 based on the SERSpeak at 900 cm1, respectively.

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Fig. 3 a Reproducibility of SERS spectra of a urine sample. b SERS spectra from artificial urine samples. Concentrations of creatinine frombottom to top are 0.5, 5.1, and 10.2 g/ mL respectively.

Table1 Creatinine concentration of urine samples from diabetic pa-tients and healthy samples, and Raman data collection parameters areshown.

Patient ID

Standard

creatinineconcentration

mg/dLScan times

10 s/ scan

Laserpower

%

CR24 M, diabetes type 1 126.3 8 5

HD46F, diabetes type 2 100.1 8 5

HK17 F, diabetes type 2 65.5 1 10

MC42F, diabetes type 2 52.0 16 5

SR45 M, diabetes type 2 114.2 4 10

SV29 F, diabetes type 1 114.2 1 10

TJ008M, diabetes type 2 6.1 1 5

RN37 F, diabetes type 1 54.7 1 10

GC006F, diabetes type 2 73.0 1 10

ML36 M, diabetes type 2 158.1 1 10

MB38 F, diabetes type 2 41.8 16 5

MR53healthy control 173.0 1 10

AS19 healthy control 99.3 1 5




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3 Experimental

3.1 Materials

All chemicals were of ACS reagent grade and were used as-

received. Deionized water of18.1 M from a BarnsteadNanopure Diamond dispenser was used for all experiments.Polychloro-p-xylylene PPX-Cl films were prepared fromdichloro-2.2paracyclophane DCPC, purchased fromParylene Distribution Services Katy, Texasand deposited on

p-type Si 100 wafers Wafernet, Incorporated, San Jose,California. Diazymes enzymatic creatinine assay kits werepurchased from Diazyme Laboratory Poway, California.Urine samples were collected from 11 diabetic patients andtwo healthy persons under a protocol approved by the Institu-tional Review Board of the Pennsylvania State Hershey Medi-cal Center, and stored in 80 C before testing.

3.2 Nanostructured Polychloro-p-xylyleneFilmPreparation

Silicon wafers were first washed in deionized water and driedunder nitrogen flow, and then added into a 1:1-v/v solution ofHCl and methanol. After 30 min, silicon wafers were re-moved and sonicated in deionized water for 10 minand driedunder nitrogen flow. The silicon wafers were then kept in

concentrated sulfuric acid for another 30 min, after whichthey were sonicated again in deionized water for 10 min. Af-ter thoroughly dried under nitrogen flow, the silicon waferswere immersed into a toluene solution containing 1% allyltri-methoxysilaneGelest, Pennsylvania and 0.1% acetic acid atroom temperature to form a self-assembled monolayer SAMon their surfaces. The silicon wafers were removed from the

solution after 60 min, sonicated in anhydrous toluene for10 min, and then dried under nitrogen flow. The silicon wa-fers were heated on a hot plate at 150 C for 4 min to bindthe SAM onto the silicon surface. Nanostructured PPX-Cl

films were deposited onto the wafers using 0.3 g of DCPC.The vaporizer and the pyrolysis chamber temperatures were

maintained at 175 and 690 C, respectively. The angle be-tween the substrate and the flux was held at 10 deg.

3.3 Metal Deposition

The preparation of substrate for SERS experiments wasstarted from the structured PPX-Cl film templates. The silverwas thermally deposited from resistively heated tungsten andtantalum boats onto the surface at about 1108 Torr basepressure in a cryogenically pumped deposition chamber.

3.4 Scanning Electron Microscope

High-resolution SEM images of the metallized PPX films

were obtained using a field emission scanning electron micro-scopeFesem, JEOL 6700F, Japanoperated at3-kV acceler-ating voltage.

3.5 Surface Enhanced Raman SpectroscopyMeasurements

Renishaw inVia microRaman equipment Renishaw, Glouces-tershire, United Kingdom was used for studying the SERSsubstrate. The instrument consisted of a 35-mW HeNe laser632.8 nm as the source, a motorized microscope stagesample holder, and a CCD detector. Raman spectra were col-lected at 5 to 10% laser power. The motorized microscopestage allowed SERS maps of the surface to be formed. The

instrument parameters were 50 objective and 10-s acquisi-tion time. 1 to 16 scans were used to obtain high signal-to-noise ratios SNRs if needed. The data are plotted withoutfiltering or smoothing in all figures. The surfaces of SERSsubstrates were treated by a UV-Ozone cleaner for 2 min,then5 Lof a urine sample was added onto the substrate anddried in air. No dilution or pretreatments were needed for theurine samples.

3.6 Urine Collection

Urine samples from patients were collected at the nephrologydepartment of Penn State Medical School under a designated

0 20 40 60 80 100 120 140 160 180 200

0

20

40

60

80

100

120

140

160

180

PredictedConcen

tration(mg/dL)

Reference Concentration (mg/dL)

0 20 40 60 80 100 120 140 160 180

0

5000

10000

15000

20000

25000

30000 SERS Peak at 900 (cm-1

)

SERS Peak at 840 (cm-1

)

SERS

Peak

Intensity

Creatinine Concentration (mg/dL)

(a) (b)

Fig. 4 a Creatinine concentration of 13 clinical urine samples and corresponding SERS peak area at 900 and at 840 cm 1 are plotted. bCreatinine concentration of 13 clinical urine samples from SERS and as enzyme-based method are plotted. Open and closed circles denote SERSpeak at 840 cm1 correlation coefficient equals 0.907 and at 900 cm1 correlation coefficient equals to 0.968, respectively.




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Institutional Review Board IRB. Artificial urine was pre-pared using 36.4 g of urea, 15.0 g of sodium chloride, 9.0 gof potassium, chloride and 9.6 g of sodium phosphatemonobasic, monohydrate dissolved in 1.5 L of water.

4 ConclusionWe use SERS to quantitatively measure the creatinine level of

urine samples from diabetic patients and healthy persons us-ing our metalized nanostructure parylene PPX-Cl film as aSERS substrate. The reproducibility and robustness of ourSERS substrate are demonstrated at various ion concentra-tions. The advantage of these new types of substrates is thatno template or lithography is involved, thus providing asimple, inexpensive, and quick production method to achievehighly sensitive and spatially uniform signals. We demon-strate a highly sensitive i.e., concentration as low as

0.5 g /mL and reproducible i.e., signal variation less than5% substrate that can be used in clinical measurements.Rapid data analyses i.e., 10-s integration time at low laserpower i.e., 2.5-mW laser at 632 nm can be performed todetermine creatinine concentration in urine. These advantages

may improve the feasibility of using SERS as a fast, effective,and inexpensive tool for urine analysis in hospitals. We shouldalso note that these data were collected at 2.5-mW laserpower and10-s integration time, which can be preferable forintegration of these substrates into a low-power handheld di-agnostic device.

Our method is also applicable to blood creatinine levelmeasurements. The normal creatinine level in blood is about

0.5 to 1.2 mg /dl,16 which can be detected by our SERS sub-strate. However, a preseparation e.g., centrifugation and fil-tration may be required to remove the interfering constitu-ents. We will focus on the feasibility of selective biomarkersand metabolites in blood in the future.

AcknowledgmentsThis research is supported by a Young Investigator ProgramAward from the Office of Naval Research, and a seed grantfrom the Huck Institute Life Sciences at Penn State. We thankDavid Allara for Raman spectroscopy access in his laboratory.

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