Detergency of stainless steel surface soiled with human brain homogenate: an XPS study

www.elsevier.com/locate/apsusc

Applied Surface Science 240 (2005) 204–213

Detergency of stainless steel surface soiled with

human brain homogenate: an XPS study

M. Richarda,c,*, Th. Le Mognea, A. Perret-Liaudetb, G. Rauwelc,J. Criquelionc, M.I. De Barrosa, J.C. Cetred, J.M. Martina

aEcole Centrale de Lyon, UMR 5513 LTDS, 69 134 Ecully, FrancebHopital Neurologique de Lyon et INSERM U512, 69 394 Lyon, France

cLaboratoires ANIOS, 59 260 Lille-Hellemmes, FrancedUnite d’Hygiene et d’Epidemiologie, Hopital de la Croix Rousse, 69 317 Lyon, France

Received in revised form 17 June 2004; accepted 17 June 2004

Available online 19 August 2004

Abstract

In the detergency field of re-usable medical devices, a special attention is focused on the non conventional transmissible

agent called prions which is a proteinaceous infectious agent. Few cleaning procedures are effective against prions and few

techniques are available to study cleaning effectiveness with respect to proteins in general. In our study, X-ray photoelectron

spectroscopy (XPS) has been used to evaluate the effectiveness of detergent formulations to remove proteins from stainless

steel surface soiled with a brain homogenate (BH) from human origin. Our results showed that XPS is a reliable surface

analysis technique to study chemical species remaining on surface and substrate properties after cleaning procedures. A

semi-quantitative evaluation of the detergency effectiveness could also be performed.

# 2004 Elsevier B.V. All rights reserved.

Keywords: Detergency; Proteins; XPS; Stainless steel; Medical device; Human brain homogenate

1. Introduction

The pre-treatment and the cleaning of re-usable

medical devices are essential and must be effective

before disinfection or sterilization. These two suc-

cessive stages of medical devices treatment aim to

* Corresponding author. Tel.: +33 4 72 18 62 67;

fax: +33 4 78 43 33 83.

E-mail address: [email protected] (M. Richard).

0169-4332/$ – see front matter # 2004 Elsevier B.V. All rights reserved

doi:10.1016/j.apsusc.2004.06.090

decrease, at the same time, the macroscopic (tissues

debris) and microscopic (bacteria, virus, etc.) contam-

inations. Their goal is to protect staff, the environment

and to support the effectiveness of the secondary

treatments of disinfection or sterilization. They entail

washing phase in which detergents and/or enzymatic

cleaners are employed.

So far, the main concern was the eradication of

microbial contaminations which are responsible for

nosocomial infections [1]. However, a new risk factor

.

M. Richard et al. / Applied Surface Science 240 (2005) 204–213 205

Table 1

Qualitative composition of detergent formulations

Formulations Qualitative composition

Hexanios G + R Biguanide

Quaternary ammonium chloride

Non ionic surfactants

has been added with the emergence of a new protei-

naceous infectious agent called prions [2] and for

which few sterilization methods are effective [3–6].

Indeed, prions are extremely resistant to conventional

gaseous disinfectants such as ethylene dioxide and for-

maldehyde, and to physical processes such as dry heat,

ionising, UV, microwaves, radiation and especially

steam heat at 121 8C for 15 min. The only effective

physical process is steam heat at 134 8C for 18 min.

Chemical disinfectants such as alcohols, ammonia,

glutaraldehyde and formalin are also ineffective. The

two main effective reagents are sodium hydroxide 2 M

and sodium hypochlorite 20,000 ppm for 1 h at room

temperature. However, these reagents are not without

effect on substrate such as aluminium or stainless steel.

Few techniques are available to evaluate deter-

gency effectiveness with respect to proteins [7]. The

main study recommended the modified orthophtalde-

hyde (OPA) method for the detection of amino acids

on medical device following cleaning. However, this

method does not take into account the medical device

itself after cleaning. In recent years, X-ray photoelec-

tron spectroscopy (XPS) is widely used for studying

protein adsorption in biomaterials area [8–14]. This

surface analysis technique makes it possible to give

the elemental composition of the outermost 10 nm of a

surface, but can also provide insights into the chemical

bonding.

The aim of our study is to show that XPS analysis is

an usable and reliable technique to study the cleaning

effectiveness especially with respect to proteins. Our

work has been carried out on stainless steel substrates

soiled by a human brain homogenate (BH). Soiled

substrates were cleaned in manual conditions with five

various detergent formulations.

(fatty alcohol ethoxylates)

Salvanios pH 10 Quaternary ammonium

Guanidinium acetate


Aniosyme PLA II Quaternary ammonium chloride


Enzymatic mixture

Aniosyme DD1 Quaternary ammonium

Biguanide

Amphoteric surfactant

Enzymatic mixture

Aniosyme N2 Amphoteric surfactant

Enzymatic mixture

2. Experimental

Square samples (1.5 cm � 1.5 cm) of austenitic

stainless steel (AISI 304) were purchased from Good-

fellow. These stainless steel samples contain boron

nitride (BN) which does not seem to be homogeneously

distributed in substrate. Its origin could come from the

elaboration of stainless steel sheets. The presence of

boron nitride makes it possible to study the cleaning

effectiveness with respect to proteins specifically, since

nitride component can be used as N1s peak internal

standard. Indeed, the binding energy of nitride chemical

bond is sufficiently different from that of amide bonds

in proteins to be easily distinguished.

Before being soiled, samples were ultrasonically

cleaned in dichloromethane, n-heptane, acetone and

n-propyl alcohol for 5 min each. Two rinses in distilled

water for 5 min under ultrasound were finally per-

formed.

Human brain tissue was homogenised at 10% (w/v)

in 5% glucose. Nuclei, blood vessels, unbroken cells

and tissues debris were sedimented by a 5 min cen-

trifugation at 1000 � g. The pellet was discarded and

the supernatant (BH) was used as model soil solution.

Five detergent formulations, supplied by Labora-

toires ANIOS, have been tested to clean soiled sub-

strates. Qualitative compositions for each formulation

tested are given in Table 1. Briefly, Hexanios G + R

(noted G + R on figures), Salvanios pH 10 (pH 10),

Aniosyme PLA II (PLA II) and Aniosyme DD1 (DD1)

formulations have detergent and anti-microbial acti-

vities at once. Aniosyme N2 (N2) has only a detergent

activity. Aniosyme PLA II, Aniosyme DD1 and

Aniosyme N2 are enzyme-containing formulations.

Salvanios pH 10 and Aniosyme PLA II are alkaline

detergents whereas Hexanios G + R, Aniosyme DD1

and Aniosyme N2 are neutral. Before being used, each

detergent solution was diluted at 1:200 in hard water

(total hardness (TH) value = 40).

M. Richard et al. / Applied Surface Science 240 (2005) 204–213206

Stainless steel substrates were soaked for 60 min in

BH solution under 3D agitation, and horizontally dried

at ambient air during 60 min. The manual cleaning

procedure involved the following steps: soiled sub-

strates were cleaned with detergent formulations for

15 min under 3D agitation, rinsed with hard water

(TH value = 19) for 2 min and vertically dried at

ambient air.

For comparison, control substrates were immerged

in detergent formulation without previous soiling and

were analysed by XPS before and after rinsing. Drying

was performed in a horizontal position before rinsing

to have a larger thickness layer of detergent formula-

tions. Drying was performed in a vertical position after

rinsing.

The XPS spectra were obtained on a Thermo VG

Escalab 220i spectrometer equipped with an unmo-

nochromatized Mg Ka X-ray source (VG XR2). The

analysed area was about 1 cm2.

Fig. 1. Survey scans of detergent formulation-expose

For each sample, a survey spectrum (0–1100 eV)

was recorded with a X-ray power of about 200 Wand a

pass energy of 100 eV. For a better legibility, survey

spectra were displayed from 0 to 730 eV. High resolu-

tion spectra of C1s, O1s, N1s, Cr2p and Fe2p were

obtained by applying a pass energy of 40 eV. The

calibration of the binding energy scale was made by

setting the C–(C,H) component of the C1s peak to

284.8 eV. High resolution spectra were curve fitted

using the Thermo VG software Avantage. The peak

area was determined using a Shirley type non-linear

background subtraction and a Gaussian/Lorentzian

(60/40) function. In the first step of curve fitting,

binding energy gaps between components of a given

peak were fixed. The full-width half-maximum

(FWHM) of all the components of a given peak

was also set equal to the value of the best-resolved

one. FWHM obtained for each sample was slightly

different according to the influence of sample charging

d AISI 304 substrates after water evaporation.


effect. The cross-section Scofield factors have been

used for quantitative analysis.

Fig. 2. N1s high resolution spectra of detergent formulation-

exposed AISI 304 substrates before hard water rinsing.

3. Results and discussion

3.1. XPS spectra of detergents

First, XPS analyses were performed on detergent-

deposited samples before the stage of rinsing and

after water evaporation to provide information about

the chemical composition of detergent formulations

(Fig. 1).

The reference sample was AISI 304 substrate

(Fig. 1: Sub) cleaned as described above and consisted

mainly of iron, chromium, carbon, oxygen, nitrogen

and boron. No significant charge effect was observed.

The binding energy of 2p level of chromium (Cr2p)

and 1s level of oxygen (O1s) (data not shown) were

related with chromium oxide passivation layer, char-

acteristic of stainless steel surface. The 1s level

binding energy of carbon (C1s) was typical of adven-

titious carbon found on air-exposed stainless steel

[10]. The presence of some boron nitride percents on

the surface were showed by the binding energy values

of N1s (398 eV) and B1s (190.5 eV) peaks (data not

shown). The origin of BN is unknown but could come

from the elaboration of stainless steel sheets. Boron

nitride is chemically inert and not water soluble.

Moreover, the binding energy of nitride chemical

bond is definitely different from that of nitrogen

chemical bonds in proteins. Therefore, it has been

used as a substrate characteristic peak and especi-

ally as reference peak to evaluate the detergent

formulations performances with respect to proteins

specifically.

XPS spectra of detergent formulations (Fig. 1)

contain mainly carbon, oxygen and nitrogen in accor-

dance with the surfactant chemical formula. A charge

effect was observed and corrected by setting the

C–(C,H) component of the C1s peak to 284.8 eV.

The substrate characteristic peaks (Cr2p and B1s)

disappeared under the detergent-formed layer, except

for Aniosyme N2 where substrate peaks were still

detected. These results show a detergent layer thick-

ness of at least 10 nm according to XPS analysis

depth. Under these conditions, XPS collected data

come especially from the detergent formulation layer.

In addition, all detergent exposed surfaces con-

tained sodium, calcium and traces of chlorine and

sulphur. Sodium has come from detergent solutions

since many compounds are sodium salts. Chlorine

could come from hard water and detergent solution

at once whereas calcium has come from hard water.

Origin of sulphur, in sulphate form (169 eV), is not

clearly identified but could come from hard water (TH

= 40) used for dilution of the detergent formulations.

Aniosyme PLA II and Aniosyme DD1 exposed sub-

strates also contained phosphorus since sodium

tripolyphosphate and phosphoric acid have gone into

Aniosyme PLA II and Aniosyme DD1 detergent for-

mulations, respectively.

Some molecules entering in the detergent formu-

lations are nitrogen-containing molecules such as


quaternary ammonium, biguanide or guanidinium. N1s

high resolution spectra performed on detergent

exposed-substrates make it possible to distinguish

detergent nitrogenized molecules from substrate nitride

(Fig. 2). The N–C (399.9 eV), N=C (400.0 eV) and

ammonium (402.2 eV) components from nitrogen-con-

taining molecules can also be accurately identified on

N1s high resolution spectra. An additional component

is slightly detected for Aniosyme PLA II, Aniosyme

DD1 and Aniosyme N2 at 400.4 eV corresponding to

amide (O=C–N) component. This result is in agreement

with the presence of enzymes in these formulations.

XPS data are in good agreement with the chemical

compounds of formulations.

3.2. Rinsing effect on detergent removal

from the surface

XPS analyses were also performed on detergent-

deposited samples after hard water rinsing to know

whether detergent formulations remained on or inter-

acted with surfaces (Fig. 3). First of all, an experiment

was performed to know whether dried detergent layer

is more difficult to remove from the surface than a

detergent layer before drying. The experiment’s

Fig. 3. Survey scans of remaining detergents on A

results show that in both cases, some chemical species

remained on surface but with less extent when surface

is rinsed immediately after exposure to detergent

formulation (data not shown). In the same way,

removed chemical species by the rinsing are removed

from the surface even after drying.

The reference substrate (Sub + R) spectra after

rinsing is similar to that of before rinsing suggesting

that clean stainless steel substrate is not contaminated

by hard water elements such as calcium and chlorine.

On rinsed detergent-exposed substrates, Cr2p and B1s

signals reappeared, indicating a decrease of the thick-

ness layer formed by detergent solutions. The elements

such as sodium, calcium and chlorine were no more

detected except for Aniosyme PLA II and Aniosyme

DD1 exposed substrates. Non-removal of calcium and

phosphorus suggests that these elements were trapped

within the remaining detergent-formed layer. The layer

thickness is estimated to at least 10 nm since substrate

characteristic peaks were not detected yet.

The cleanest surface after detergent exposure and

rinsing was Aniosyme N2 formulation exposed sur-

face.

However, the intensity of the Cr2p and B1s signals

remained well below the one of reference substrate

ISI 304 substrates after hard water rinsing.


Fig. 4. N1s high resolution spectra of detergent formulations

remaining on AISI 304 substrates after hard water rinsing.

suggesting that the surface could be contaminated by

some chemical species contained in the formulations

such as non ionic surfactants. Indeed, many detergent

formulation molecules consist of carbon, oxygen and

hydrogen and C–(C,H) component cannot be distin-

guished from C–(C,H) of adventitious carbon. The

remaining chemical species cannot be identified in

the unquestionable way by XPS. However, it is not a

restrictive data since nitrogenized molecules only,

and especially proteins are taken into account in our

study.

N1s high resolution spectra of formulations

exposed substrates showed tendency of nitrogenized

surfactants to remain on surfaces even after rinsing

(Fig. 4). The NNitride/NTotal ratio was calculated to

evaluate hard water capacity to remove detergent for-

mulation molecules from the surface (Table 2). For

Hexanios G + R, Salvanios pH 10 and Aniosyme DD1

exposed substrate, NNitride/NTotal ratios are well below

that of reference substrate (0.823). This result showed

that surfactant molecules of detergent formulations,

adsorbed on substrate, were not desorbed much by hard

water rinsing. However, Aniosyme PLA II and Anio-

syme N2 seem to be easily removed by rinsing since

the ratios, 0.85 and 0.83, respectively, are closed to the

reference (0.83). These data could be explained by

formulations complexity. Indeed, Aniosyme PLA II

and Aniosyme N2 formulations consisted of only one

N-containing molecule, quaternary ammonium chlor-

ide and amphoteric surfactant, respectively, whereas

Hexanios G + R, Salvanios pH 10 and Aniosyme DD1

consisted of two at least N-containing molecules of

which one is biguanide or guanidinium. These mole-

cules are C=N and C–N containing molecules and they

seem to partially remain on stainless steel surface

(Hexanios G + R, Salvanios pH 10 and Aniosyme

DD1 on Fig. 4). For all ammonium-containing formu-

lations, it is interesting to note that adsorption of

ammonium was almost completely reversible since

no component was resolved at 402.2 eV.

Table 2

Ratios Nnitride/Ntotal

Nnitride/Ntotal

Hexanios G + R Salvanios pH 10

Formulation 0.47 0.09

Formulation + rinsing 0.59 0.41

Therefore, a strong interaction between the stain-

less steel surface and some nitrogenized chemical

species of the detergent formulations could be

expected.

3.3. XPS analysis of brain homogenate soiled

substrate

To study the cleaning effectiveness in the most

realistic conditions, brain homogenate has been used

Aniosyme PLA II Aniosyme DD1 Aniosyme N2

0.54 0.39 0.83

0.86 0.57 0.83


Fig. 5. Survey scans of AISI 304 substrate and brain homogenate soiled AISI 304 substrate before (BH) and after hard water rinsing

(BH + R).

as biological soiling. Indeed, brain homogenate soiled

stainless steel substrate may be a good mimic of soiled

medical device following a neurosurgical interven-

tion. XPS analysis of human brain homogenate soiled

substrate is shown in Fig. 5 (BH). The control assay

was soiled substrate rinsed with the hard water (TH

value = 40) used to dilute detergent formulations

(Fig. 5: BH + R). BH exposed substrates contained

mainly carbon, oxygen and nitrogen since brain homo-

genate is made up of proteins, lipids and polysacchar-

ide mixture. Nitrogen is present in peptidic bonds and

in some amino acids side chains of proteins.

The residual layer was thick since iron, chromium

and boron were not detected. The hard water rinsing of

soiling (BH + R) seems to slightly decrease thickness

layer since B1s started being detected. Cr2p was not

detected yet. This result may be explained by a larger

boron 1s electrons mean free path compared with

chromium 2p electrons. Indeed, boron kinetic energy

is larger than that of chromium in XPS. Therefore,

boron electrons come from deeper. The other hypoth-

esis would be a non-homogeneous steel surface with

some BN grains which could emerge from the remain-

ing brain homogenate formed-layer. Some other ele-

ments such as calcium and chlorine have also been

detected on surface and have certainly been brought

by hard water.

C1s and N1s high resolution spectra of soiled

substrate before and after rinsing highlight the com-

ponents modification following the rinsing (Fig. 6).

The main component of the brain homogenate C1s

peak is C–O. This component come from the 5%

glucose buffer used to homogenise human brain tissue.

This component significantly decreased after hard

water rinsing since glucose is a soluble compound.

The amide component also slightly decreased with

rinsing. The decrease of the amide component is

observed after rinsing on both C1s and N1s peaks.

The N1s peak of the soiled and rinsed substrate (BH +

R) showed a prevalence of the N–C=O component

suggesting a great protein adsorption from the brain

homogenate. N–C component is assigned to amino

acid side chains. These results show the weak deter-

gent capacity of hard water with respect to proteins.


Fig. 6. C1s and N1s high resolution spectra of reference sub-

strate (Sub), soiled substrate (BH) and soiled and rinsed substrate

(BH + R).

3.4. Cleaning of brain homogenate soiled substrate

In order to study detergent effectiveness, soiled

substrates have been cleaned with the various formu-

Fig. 7. Survey scans of brain homogenate soiled AISI 304

lations and rinsed with hard water (TH value = 19).

Survey spectra of cleaned substrates are shown in

Fig. 7. Substrates which have been cleaned by deter-

gent formulations seemed to be cleaner than the con-

trol one (Fig. 7: BH + R) since chromium and boron

are apparent on deterged substrates. Calcium and

phosphorus remained on Aniosyme PLA II-cleaned

substrates. Broadly, the cleaned substrates are far from

being as clean as the reference substrate, since many

C, O and H containing molecules could remain on

surface.

N1s high resolution spectra of soiled and cleaned

substrates (Fig. 8) highlighted the effectiveness

of detergent formulations to remove proteins from

stainless steel surface even if the removal was not

complete. The formulation performances for the

cleaning of soiled substrates are evaluated thanks to

the NNitride/NAmide (Table 3). Aniosyme N2 is the best

detergent formulation to remove proteins from stain-

less steel surface since the ratio value is the highest.

The other formulations seem to be effective against

proteins even if N–C=O component is still detected.

However, Aniosyme PLA II and Aniosyme DD1 are

substrates cleaned with the detergent formulations.


Fig. 8. N1s high resolution spectra of brain homogenate soiled AISI

304 substrates cleaned with the detergent formulations.

enzyme-containing formulations. Although enzyme

concentration in formulation is low, the difference

between the two protein sources cannot be made by

XPS analysis.

These results suggest strongly that stainless steel

substrates are contaminated by some detergent mole-

cular species after protein removal.

Table 3

Ratios Nnitride/Namide for soiled substrates deterged and rinsed

Soiled substrates Nnitride/Namide

BH + R 0.49

Hexanios G + R 7.14

Salvanios pH 10 5.26

Aniosyme PLA II 9.09

Aniosyme DD1 6.25

Aniosyme N2 33.3

4. Conclusions

XPS has been used to study detergency effec-

tiveness on stainless steel surface specifically with

respect of proteins, in the most realistic possible

conditions. From a technical point of view, this work

shows that XPS is a useful and reliable technique since

remaining nitrogenized chemical species on surface

can be studied. XPS makes it possible to study the

surfactant adsorption reversibility on surface and to

perform a semi-quantitative evaluation of detergency

effectiveness.

From detergency point of view, our results show

that tested detergent formulations are broadly effec-

tive in protein removal even if it is not complete and

that some chemical species remain on surfaces. How-

ever, further investigations will be carried out by ToF-

SIMS to get molecular information about chemical

species remaining on surfaces and their chemical

interaction with the stainless steel surface.

From prion point of view, it would be interesting to

study the residual infectivity remaining on substrate

after cleaning. Indeed, our results raise the question

of the infectious minimal quantity. Is the protein

quantity remaining on cleaned surfaces sufficient to

be considered as potentially at risk?

Acknowledgements

This work was supported in part by GIS ‘‘Infections

a prions’’ projet F97.

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Detergency of stainless steel surface soiled with human brain homogenate: an XPS study