Two methods for eliminating luminol interference materials are described. One method eliminates interference from organic material by pre-reacting a sample with dilute hydrogen peroxide. The reaction rate resolution method for eliminating inorganic forms of interference is also described. The combination of the two methods makes the luminol system more specific for bacteria. Flow system designs for both the firefly luciferase and luminol bacteria detection systems are described. The firefly luciferase flow system incorporating nitric acid extraction and optimal dilutions has a functional sensitivity of 3 x 100,000 E. coli/ml. The luminol flow system incorporates the hydrogen peroxide pretreatment and the reaction rate resolution techniques for eliminating interference. The functional sensitivity of the luminol flow system is 1 x 10,000 E. coli/ml

Thomas, R. R.

English

NASA Technical Reports Server

General Disclaimer 
One or more of the Following Statements may affect this Document 
 
 This document has been reproduced from the best copy furnished by the 
organizational source. It is being released in the interest of making available as 
much information as possible. 
 
 This document may contain data, which exceeds the sheet parameters. It was 
furnished in this condition by the organizational source and is the best copy 
available. 
 
 This document may contain tone-on-tone or color graphs, charts and/or pictures, 
which have been reproduced in black and white. 
 
 This document is paginated as submitted by the original source. 
 
 Portions of this document are not fully legible due to the historical nature of some 
of the material. However, it is the best reproduction available from the original 
submission. 
 
 
 
 
 
 
 
Produced by the NASA Center for Aerospace Information (CASI) 
https://ntrs.nasa.gov/search.jsp?R=19760021720 2020-03-22T14:42:45+00:00Z
1^0EIi G nLIXOSf ACE CONIWAN1	 `io ton Te"'Houston, Texas 77058
A Oivisiou of The flovinj Cumtiany
May 10, 197G
5-2510-11011-2-1463
National Aeronautics and Space Administration
Goddard Space flight Center
Greenbelt, Maryland 20771
Attention:	 Rhoda Schwartz
Contracting officer/287.1
Subject:	 Contract NAS5-22545
Transmitted herewith is the Quarterly Report for the subject contraLA , covering
the period January - March 1976.	 1
a
If there are any questions, please contact the undersigned at (713) 488-0910,
extension 209.
PACz
DIVISION
Robert S. Anderson
iiouston Contracts i'tan;:,ger
cc/w. enclosure:
Publication Branch/251
Patent Counse1/204
Technical Officer (7)/726
	
' ^I	
~'^
OL i
R,^	 ^^,C \ EPA\,
(NASA-CR-'44768)	 TWO TECHNIQUES FOR
	 N76-28808
ELIMINATING LUNINUL INTERFERENCE MATERIAL
	 11c	 3.50
AND FLOW SYS: EM CONFIGUF.AITO III
 FOR LUMINOL
AND FIFFFLY LUCIFERASE SYST'7MS Quarterly
	 Unclas
ve p ort, Jan. - Mar. 1976 (boeina Co..
	 G3/51 48127
Enclosure to 5-2510-IIOU-2-1483
dated May 10, 1976
Two Techniques for Eliminating Luminol Interference Material and
Flow System Configurations for Luminol and Firefly Luciferase Systems
Prepared by:
RICHARD R. TI1014AS
The Boeing Company
P. 0. Box 58747
Houston, TX 77058
Approved by:
ROBERT Ii, WUSS
The Boeing Company
P. 0. Box 58747
Houston, TX 77058
Quarterly Report for Period January - March 1976
Prepared for
Goddard Space Flight Center
Greenbelt, MD 20771
ti
r
L. .
n
^. Report Ito, 2. Government Accession No. 3. Reeipleni's Catalog No,
2
77Y J a an - a I 1 a Two T	 li-514Ueufo7;ififfi Ming —rTtepml ale	 ,
LUminOl Interforence Material and Flow
System Configurations for Luminol and 6. Performing Organlsallon Code
h'' -efl.v r,uci.fel."asn_Sy_stcrns
B. Performing Orgonlrellon Report No.7. Authors)
Hichard R. Thomas
9. Performing Organisation Name and Address 10. Work Unit No,
The Boeing Company
11. Contract or Grant No.P.O. BOX 5870
tloustol .	 TX	 7,70583 NAS 5-22545
13. Type of Report and Period Coyarcd
12. Sponsoring agency Name and Address
NASA/Goddard Space Plight Center
II -January-March
1976
Grace Lee Picciolo, Code 726 1 4. Sponsoring Agency Coda
Greenbelt, MD	 20771
15. Supplementary Notes
a
16. Abstract
Two methods for eliminating luminol interference materials
are described.	 A method has been developed which eliminates
interference from organic material. 	 By pre-reacting a sample
with dilute hydrogen peroxide organic materials can be in-
activated.	 The reaction rate resolution method for eliminating
inorganic forms of interference is also described. 	 The com-
bination of the two methods ma)ces the luminol system more
specific for bacteria.
Plow system designs for both the firefly luciferase and luminol
bacteria detection systems are described. 	 The firefly luci-
ferase flow system incorporating nitric acid extraction and
optimal dilutions has a functional sensitivity of 3 x 10 5 L. Soli/
ml.	 The luminol flow system incorporates the hydrogen peroxide
pretreatment and the reaction rate resolution techniques for
eliminating interference.	 The functional. sensitivity of the
luminol flow system is 1 x lOd P. Soli/ml.
17. Key Words (Selected by Author(s)) 18. Distribution Statement
Luminol, Interference,
Luminol-Luciferase Plow System
19. security Classlf. (of this report) 20, Security Clossif. (ol this poga) 21. No, of Pages
	
122. Pries
UNCLASSIPIliD UNCLASSIFIED
1	
' Por rata by the Nn  Tonal Technlani I [if OrIan1I411. Service, Springfield; Virginia 22151.
iz
1 e
YTABLES
Page 110.
Table I. Effect of 0.5% hydrogen peroxide 5
pretreatment (10 minutes) on
luminol oxidizing agents.
Table II. Effect of 0.5% hydrogen peroxide B 1
pretreatment (10 minutes) on bacteria,
Table III. Effect of buffered 0.5% hydrogen B
peroxide pretreatment on catalasc
and E. coli.
Table IV. Luminol response following inter- 9
o ference elLnination techniques.
1
i
^i
I
1
i
I
t 4
ILLUSTRATXONS
Figure 1.
Figure 2
Fis"re 3
Figure 4.
Figur, • 5
Figure G.
Figure 7.
Figure 8
P,e No.
Peal; luminol light responsr, to 	 4
1 x 10- 9 M catalase with varying
hydrogen peroxide concentrations.
Effect of 11302 concentration for
	
G
hydrogen peroxide pretreatment of
catalase (4.4 x 10-7 '1,1).
Effect of• 11202 concentration for
	
7
hydrogen peroxide pretreatment• of
E. coli..
Reaction rate curves for various 10
luminol oxidizing agents (arbitrary
concentrations).
Automated luminol flow system in- 12
corporating hydrogen peroxide pre-
treatment and reaction rate resolu-
tion for eliminating interference.
Peal; light  response from various 13
concentrations of washed E. coli
using the luminol flow system.
Automated firefly luciforase flow 14
system for detecting bacterial
ATP including nitric acid extrac-
tion and subsequent dilution.
Light response from various con- 15
centrations of washed E. coli
using the firefly luciferase flow
system.
- 2 -
I. Hydrogen Peroxide
It was reported in Report No. 11 that sodium perborate produced
a higher peak light• response from the luminol reaction than did
hydrogen peroxide. It has since been found that on a mole per
mole basis, of all the hydrogen peroxide producing compounds,
sodium perborate, sodium peroxide as wall as hydrogen peroxide
all produce the same light response for luminol oxidizing agents.
Figure 1 shows that the peak light response from 3uminol for a
porphyrin sample is a function of hydrogen peroxide concentration.
Although the peak light response varies with hydrogen peroxide
concentration, the total light from a particular sample remains
constant. Because of this, the shape of the curves for various
peroxide concentrations vary from a sharp, high peak at high
peroxide concentrations to lower but broader curves at lower
concentrations. This combined with the characteristic reaction
rate ca vas for the vari.oras luminol oxidizing agents provide
the basis for the reaction rate resolution method for eliminating
interference.2
of the three hydrogen peroxide producing compounds used in this
laboratory, sodium perborate and sodium peroxide have the ad-
vantage of being relatively stable solids. sodium perborate
has the disadvantage of having a limited solubility in water,
0.1625M = 0.55;. Sodium peroxide is vo7;y soluble in water;
however, in addition to producing hydrogen peroxide when dis-
solved, also produces sodium hydroxide. This limits its use
as a pretreatment since the sodium hydroxide could damage the
bacteria cells. Although it has a limited shelf-life, hydrogen
peroxide is still the most convenient reagent to use for the
luminol mixture. it is recommended that the luminol-peroxide
reagent mixture not be used when over four hours old.3
- 3 -
REPRODUCIBILITY OF THE
ORIGINAL PAGE I6 POOR
1
'4
Flow
Figure 1.	 Peal. liuni nul li f ,,ht rer pons
hydroi;cn peroxicl( ,
 corlcentr
] U^t
102
] 03
lW
_^^I-
Ili
I^
{1111
III
-.- l
^ 1
ll l
I;i
I l I__
_1
I CI
^^l l ^I JJ_
X 10-9
 1.1 catulare with varyi nj-,
a1
l r>'
a^
a^
.t;
n; 100
1
10_] o
G
a
J
10	 1
12	 3 4 5 6 U 1
In '	 10- p 10	 100
	10 	 10`
I':i.nal 1[y(Iro„c-, n Peroxide Conccht.ratiUn
(Per c(-nt)
- 9 -
II. hydrogen Peroxide Pretreatment for Eliminating Interference
A technique has been developed for eliminating organic luminol
interferences such as catalase, hemoglobin, and other soluble
extra-cellular porphyrins. This method involves a pre-incubation
of the sample with a dilute concentration of hydrogen peroxide
to pre-react and thus inactivate the organic interferences. The
bacterial porphyri.ns in the intact cells are proteotcd and only
react with the luminol reagent after they have been ruptured by
th> sodium hydroxide in the luminol mixture.
Table I shows the effect of 0.5% hydrogen peroxide pretreatment
on several. compounds which could interfere with the luminol-
bacteria detection system. The 10 minute pretreatment eliminated
over 9M of the interfering signal from the porphyrin materials.
Figure 2 shows the effect of hydrogen peroxide concentration and
time on a catalase sample. The greatest reduction of signal
takes ii-ace within the first ten minutes of the reaction. Con-
centrations greater than 0.1% are necessary for effective reduction
of interference signal.
TABLE I. Effect of 0.5% hydrogen Peroxide Pretreatment
(10 minutes) on Luminol Oxidizing Agents
Reduction of Peak Signal
hemoglobin	 95;
Catalase	 94%
Extracted Bacterial Porphyrins	 97'5
Potassium Ferricyanide 	 50%
Cobalt (ous) Chloride	 20%
Ferrous Sulfate	 0
Figure 3 shows tile. affect of hydrogen peroxide concentration and
time on a sample of stationary phase B. coli. At hydrogen peroxide
concentrations less than 1.5'5, no significant reduction of response
from E. soli was observed. Table 1I shows that the age Of the
cells has a definite effect on the susceptibility of the cells
to hydrogen peroxide pretreatment. Actively dividing cells such
as found in log phase growth are more susceptible to reduction
by hydrogen peroxide than cells in the stationary growth phase.
- 5 -
II I
I k I
1
I
. I
I 'T
71
	
I
I
, '	 iy
-atalcl xprut^c;a tmf. it ' of L
04 .11	 .	 .	 .	 .	 .	 .	 .	 .	 .	 .	 .	 .	 .
co	 tt.l,,n
E; 62
77
RPV.VPuc4tL4, -v Qv 'ilu,
ol"101,14AL. MGH	 POOL,,
- w
iI
. ,
c"
a,
V)
t;	 tr
c^
c,
c:
C.1
a^
r^.
I`.
lug
_
'i	 ,1.	 ...^,	 ..,...^.,r.,,	 .•..._,	 ^. r	. ....^.......^.._,... ,....._.....	 ,...,..	 i"•^.
;; ► !i ^!rir	 tli^^
i
6W.4., -. y
FTABLE II. Effect of 0.51 hydrogen Peroxide Pre tract tmenl:
(10 minutes) on Bacteria.
Reduction of Peak Signal
Growth Phase
Stationary
	 Log
Escherichia coli	 B%	 24%
Bacillis subtilis	 16%	 36%
Pseudomonas aeruginosa	 43%	 40%
Hydrogen peroxide is a more effective oxidant at higher plis.
By raising the ph of the 0.5% hydrogen peroxide pretreatment,
the pretreatment is slightly more effective in eliminating
solublr. porphyri.n interference t±1,51e at the same tisae increasing
the signal from the bacteria. Table III shows the effect- of
various buffered ph solutions of hydrogen peroxide and their
offect•
 on catalase and E. coli.. As can be seen from the table
0.5% hydrogen peroxide lds;fFered between PH 5.5 and 7.3 is most
effective in eliminating catalase while decreasing the signal
loss from E. coli. while a hydrogen pc ,-:oxide pretreatment
buffered around ph -7 is hest for eliminating porphyrin inter-
forenco and preserving the response from bacteria, the dif-
ference is not, however, great enough to warrant• bufforing
the hydrogen peroxide pretreatment on a permanent basis.
TABLE 111. Effect- of Buffered 0.5% HydrogLis Peroxide
Pretreatment on Catalase and E. coli.
Reduction of Signal After. 10 Minutes
10-814 Catalase	 E. coli
PH Buffer (log phase)
4.77 Citric acid 79% 402
5.48 citric acid 83% 27%
7.27 Phosphoric acid 83% 311;
9.19 Boric acid 87% 41%
In sununary, the 0.52 hydrogen purrxide pretreatment for a peri.od of
ten minutes is the optimal condition for eliminating organic inter-
ference. Over 90% of soluble extra-cellular porphyrins can be
eliminated using this technique.
- 8 -
I
d^
--
i r^
III. Reaction Irate Resolution
As can be seen from Tablo I, the hydrogen peroxide pretreatment
does not adequately eliminate inorganic forms of interference
for the luminol reaction.
While there was a 20% reduction of luminol signal from pneLreated
cobalt(oue,) chloride, no reduction of signal was noticed with
ferrous sulfate. The ferrous sulfate may have already been oxi.-
dizcd to ferric sulfate when put intoint  solution before assaying
explaining L•he lack of a reduction in signal. To eliminate
inorganic interferences cf the luminol-bacteria system the re-
action rate resolution method described in Repor.L No. I must be
used. 4
 Pigure 4 is an illustration of the type. of reaction rage
curves observed with several luminol oxidizing agents. When used
in conjunction with the hydrogen peroxide pretreatment, the re-
action rate resolution me;:hod can make the luminol system more
specific for bacteria deLection.
Table IV is data produced with the luminol flow system while at
the Boring Laboratory in Houston, Texas. The effect of each
inL• (is-fpronce elimination method, both individually and together,
on several water samples is shown, Overall 9B'J of the inter-
ference was eliminated from contaminated water samples.
TABLE IV. Luminol Response Hollowing Interference 1•'lianinatiou
Techniques.
Relative Light Units
P. soli
Water. purified Seeded
Deionized by Reverse Tap 1)eionized
Water Osmosis Water Water
1) 140 pretreatment 0.49 17 41 27
2) 11202 pretreatment U.40 2.1 3.5 30
3) Reaction rate resolution 0.23 0.83 2.4 37
4) Combination of 2) and 3) 0.21 0.30 0.67 49
Overall Reduction
of interference signal 57 q. 98 a; 98% (•h],81^)
- 9 -
co
_b
	
I	 ^	 r
	
`	 I
	
w	 f
	
U)
	 irk
	
-i	 O	
<
	 U
	
Q	 w^
U
N
I	 oz
r 0
-J	 w
U)
't
I
r
U	 C7	 `	 cr;
r ^
Q	 00	 ':o	 d	 LVr
:JsNlocjlK- ]H 11-101-1
— 10 —
z
r^
X
O cn
U OZ
F-
-^ z
U) lL'
D U
Cz
rr 0
U
w^
r^ cn
>CCrt Q
:^
U cn
I Li z
Lij
CC l
O
U
Q
w
u
L . ,
t..
IV. flow System configurations
A schematic diagram of the luminol flow system is shown in Figure 5.
This system incorioraL• es l:he two methods for eliminating interference
problems. 1`h: hydrogen peroxide live trea Well L• allows the sample to
pre-react the organic interferences and 0:49. hydrogen peroxide for
it period of 2 minutes. The sample then reacts with the luminol
reagent (2.5 x 10-4 M luminol, 0.11 11202 and 0.75 N Na011) for a
period of 10 second„ before reaching the photomulti.pli.cr tube.
This sLop, reaction rate resolution, eliminates the interference
from inorganic materials. The podk height is then measured to
determine the bacteria concenLration. Figure G shows the luminol
flow nystem response to E. soli. The functional. sensitivity of
the system is 1 x 104 L, coli%u11.
Tile firefly luciferase flow system for detecting bacterial ATP
has been constructed according to Pigure 7. The flow system
incorprrates the optimal extraction and assay methods while re-
quiring minimum dilution of the sample. The extraction step
requires a GO second residence time of sample with 0.1 N nitric
acid and than subsequent dilution with water. The actual assay
requires 0.2 ml A luciferase.. Pirn ;e 0 is all 	 coli concentra-
tion curve obtain>.d with the flow system using Ful'onL luciferase
(1 -vial brought up to 3 ml with 0.25M TRIS, 10- 2M 1.1gSO4 and 1.0-3M
Cleland's reagenL). The graph compares the response when measured
as peal; height and when tl,e signal is integrated for a period of
Go .seconds. Nhile the sensitivity is the same in both cases,
about 3 x 105 cells/ml, better linearity is achieved with the
int-ccratcd signet..
The sensitivity of the flow system is about• one-half. the sensitivity
of the injection system. 1 `11i.s difference liar, been attributed Lo
the configuration of the flow cell. The walls of the flow cell are
much thicker than the walls of the cell used for the injection
system and apparently absorb some of the light produced in the
reaction.
Y.
11 -
^ n^^I
Lcs^
co
.J I—
7 LL!
.D u.t
0
cc^
0
rr_
--_	 t1t
k-^	
rr
U
tlJ
l+1
111
lZ
U
Q U
tY <i
O ^ 11
U ^ Lr
Lei.
L
w LU I^
cn I-'
[n ua
LL CL
O
I11 = =:
n
O r`:(l_ <^
MilJ Z C
Q CO^.^
oo^f
I	 ^1
Q S (1.
tL
Y
111
J
[L
F: ll1
_J C1^
} h0
O
11
c
_?_
ITD
^	 c
LL
Q
tll
U '^
~-JN Cn
W
^a L
to ^;
rr: U T--
LL
Y'
1pi
O W
JU
O
- Z
W <J
L11
0
W
Q
Q
r^
12 -
cv
O
CJ
s, =
C r,
3.05	 1.06
inl	 (Coul_t(!r cotult)
e,
103
30-' 1 (
	 2_! 1!,4 0 ,
103 	101,
10`
1 U1
r^
(1 r
-lip
dAI L_IL
.107	 101,
^	 r
i ICJ CEO
}'i1;ure	 Peak 110it rcnpon-n c from variour coneentrntion. of wz,:.hed
E. col i usint , the ltrnhwl flow Cyr tcm.
- 13 -
E
?E
o ^^
0
Q
O I,
I	 1L1
O	 ^
Uf
U) LlJ
C-) C
LIJ
ry
cn	
^:
F
LI	 n	 ^'^
CC	 1	 T
Wl1J_
r i	 .
1.
"r"
•	 I
111
ul
I	 `J Il )
pU
LL	
O
f-•
Q
,-j	 ka
tl^
O.
t7_
U
U
w
l
t
U ^,
r_
11 ' O
I-
CC , U
<
w U
U)
U) z
o1`
J Fu_ = i
ulJ_
d Z L
lL
J G ,_
J Q C.
^ W f
•w 	_
C`
lU
^.L..
n
CD `^
110()
101
Fi (Cure B . Light rcrponse from var3 cnjc+ concentrations, of was hcd E. rc.1 i
usint, the i'i rct'] y luci fernre flaw syst(.m.
10 p
=
1.07
106
10	 ► ^
m
'i
to
ct	 l
fl,
I
t 4
10'I
i
N^
,V
sin
10	
^^ ...
^h
n,
10-3
d
6
s
4
S
2
10-h
10-]
0N
U
a,
4 .)	 _•
o to
1 a	 1.05
	
I()6	 1Uf
	
108
E. co1.i / 1111	 (Platte' count)
- l!) -
► 01
c
1.0 9
J
lREXERENCGS:
1. R. R. Thomas, "DevelopmenL of a Chemilumincuccnt and
Bioluminescent Systan for the Detection of Bacteria in
Wastewater Pffluent", Quarterly Report- No. 1 to NASA;
Contract No. NAS 5-22595, 1975, p. 9.
2. Tbid., p. 16.
3. Personal conununication with Dr. Norma D. Searle, 13
February 76.
9. Quarterly Report No.,l to NASA, p. 16.
0
a
r
I
1
-IG-
y
•	 44
^-'t'.tTm-.—rY^+-	 -Y..^-.^..	 ^C't.-vim.... .. a	 ,.:..^. --„^.. _...•..._,.—	 .^.._--
	
e.l	 `


Two techniques for eliminating luminol interference material and flow system configurations for luminol and firefly luciferase systems

Abstract

Similar works

Full text

Available Versions

NASA Technical Reports Server