1. Development and Evaluation of an In house Collector for Oral FluidsDevelopment and Evaluation of an In-house Collector for Oral Fluidsp
Jesús M González and R Sam Niedbala (san204@lehigh edu)Jesús M. González and R. Sam Niedbala (san204@lehigh.edu)
Department of ChemistryDepartment of Chemistry
Lehigh UniversityLehigh University
1 Abstract 4 Results In order to reduce the hydrophobicity of the Porex discs a chemical1. Abstract 4. Results In order to reduce the hydrophobicity of the Porex discs a chemical
treatment was devised The treatment consisted in submersion of the
Collection of oral fluids varies from crude to elegant devices Th t i l t t d ll l (Ult ll)
treatment was devised. The treatment consisted in submersion of the
di i b ff l ti t i i f t t D t itCollection of oral fluids varies from crude to elegant devices. Three materials were tested, a cellulose sponge (Ultracell), a discs in a buffer solution containing a surfactant. Due to its presence
As part of a larger project to simultaneously detect both
Three materials were tested, a cellulose sponge (Ultracell), a
PVA (PVA) d i id l th l di (P ) Th
discs in a buffer solution containing a surfactant. Due to its presence
th GRASE li t T 20 h t t t t ThAs part of a larger project to simultaneously detect both
t i d l i id i l fl id i l i i
PVA sponge (PVA) and a rigid polyethylene disc (Porex). The on the GRASE list, Tween 20 was chosen as treatment agent. The
protein and nucleic acids in oral fluids, a simple inexpensive
p g ( ) g p y y ( )
materials were evaluated with oral fluids spiked with GFP
, g
discs were submerged in Tween 20 solutions for a given period
p , p p
collector was designed and tested that effectively gathered
materials were evaluated with oral fluids spiked with GFP discs were submerged in Tween 20 solutions for a given period,
collector was designed and tested that effectively gathered
expressing cells at a concentration of 108 cells/ml (high
g g
followed by drying at 37°C for a specified time The influence of threesufficient analyte to detect both analyte-types. The collector expressing cells at a concentration of 108 cells/ml (high followed by drying at 37 C for a specified time. The influence of threesufficient analyte to detect both analyte types. The collector
consists of a handle and a circular holder in which a concentrations) and 103 cells/ml (low concentrations) The process variables (Tween 20 concentration treatment and dryingconsists of a handle and a circular holder in which a concentrations) and 10 cells/ml (low concentrations). The process variables (Tween 20 concentration, treatment and drying
chemically treated sintered plastic disc is placed The high concentration cell solution was measured by time) on the collecting capacity of the discs was investigated Thechemically treated sintered plastic disc is placed. The
t t t i t f b ff d f t t t d th
high concentration cell solution was measured by
fl h th l t ti l ti
time) on the collecting capacity of the discs, was investigated. The
lt h i Fi 3 d 4treatment consists of buffer and surfactant to reduce the fluorescence whereas the low concentration solution was results are shown in Figures 3 and 4.
hydrophobicity of the plastic To operate the device is placed
fluorescence whereas the low concentration solution was
d b ti l t
results are shown in Figures 3 and 4.
Th i t ti l t i Fi 4 h t d d f thhydrophobicity of the plastic. To operate, the device is placed measured by counting on agar plates. The interaction plots in Figure 4, show a strong dependency of the
in the buccal cavity. After collecting oral fluids it may be
y g g p
Cell lysing experiments were first conducted to ascertain if any
p g , g p y
amount collected by the Porex discs on the drying time Tween 20in the buccal cavity. After collecting oral fluids it may be
placed into fluids for transport eluted into other devices such
Cell lysing experiments were first conducted to ascertain if any amount collected by the Porex discs on the drying time. Tween 20
placed into fluids for transport, eluted into other devices such
reduction in cell concentration after expression was due to concentration and treatment time showed a weaker effect on the
as a chip for testing or stored for future analysis The design
reduction in cell concentration after expression was due to concentration and treatment time, showed a weaker effect on the
as a chip for testing, or stored for future analysis. The design
f th ll t t ll th ll ti di k t t sample retention or cell death As a result of these Porex performance; consequently the treatment was defined asof the collector system allows the collection disk to separate sample retention or cell death. As a result of these Porex performance; consequently, the treatment was defined asy p
from the handle In this way the pad can be effectively experiments it was determined that around 3% of the cells 0.1% Tween 20, 5-30 minutes treatment time with overnight drying.from the handle. In this way the pad can be effectively experiments it was determined that around 3% of the cells
d t th Ult ll d th P t i l di d
0.1% Tween 20, 5 30 minutes treatment time with overnight drying.
washed and the target analytes eluted. The collector disk exposed to the Ultracell sponge and the Porex material died,washed and the target analytes eluted. The collector disk
gathered on average 150 l The optimum volume ratio for
p U p g ,
bet een 15% and 20% of the cells e posed to the PVA ere
Average Saliva Absorbed with Different Porex Treatments
gathered on average 150 l. The optimum volume ratio for between 15% and 20% of the cells exposed to the PVA were
g
elution of either proteins or nucleic acids/cells was 3:1 To
p
lysed 0.18
elution of either proteins or nucleic acids/cells was 3:1. To
l t th ll t d d l i E h i hi li
lysed.
evaluate the collector pads a model using Escherichia coli Experiments were conducted using the methodology 0 16p g
cells expressing green fluorescence protein was developed
Experiments were conducted using the methodology 0.16
cells expressing green fluorescence protein was developed.
described In the case of the of the sponges samples were 0 14
Samples spiked with E. coli were used to measure recovery
described. In the case of the of the sponges, samples were 0.14
Samples spiked with E. coli were used to measure recovery
which was approximately 90% Similar results and recoveries collected until the sponges expanded to their maximum 0 12
g)
which was approximately 90%. Similar results and recoveries collected until the sponges expanded to their maximum
it ll t 60% it I th f th i id
0.12
ed(
were found when testing for total protein thus the collector capacity as well as to 60% capacity. In the case of the rigid
sorb
were found when testing for total protein, thus the collector
d t d t l th d d li th t t
p y p y g
disc samples were allowed to absorbed until saturation (60
0.1
Abs
appeared to adequately gather and deliver the target disc, samples were allowed to absorbed until saturation (60
ivaA
pp q y g g
analytes Finally to test storage stability collector discs were
p (
seconds) With high concentration samples the materials were
0.08
Sal
analytes. Finally, to test storage stability, collector discs were seconds). With high concentration samples the materials were
age
stored for up to 10 weeks at 4°C, 37°C and room flushed with a volume of PBS equal to the volume absorbed 0.06
Avera
stored for up to 10 weeks at 4 C, 37 C and room
temperature Similar to freshly made collectors it was found
flushed with a volume of PBS equal to the volume absorbed
A
temperature. Similar to freshly made collectors, it was found as well as with three times the volume absorbed In the case 0.04
that over 90% of the cells absorbed onto the collector and
as well as with three times the volume absorbed. In the case
f 3 1
that over 90% of the cells absorbed onto the collector and
90% f th t t l t i i iti ll t i th l
of low concentration samples only a 3:1 [diluent:sample] 0 02
over 90% of the total protein initially present in the samples
of low concentration samples only a 3:1 [diluent:sample]
fl hi ti d R lt h i T bl 1 d
0.02
p y p p
tested were recovered after elution Also after 10 weeks of flushing ratio was used. Results are shown in Table 1 and 0tested were recovered after elution. Also after 10 weeks of g
Figure 2
0
0 1%Tween- 0 1%Tween- 0 5%Tween- 0 1%Tween- 0 5%Tween- 0 1%Tween- 0 1%Tween- 0 5%Tween- 0 1%Tween- 0 5%Tween-
storage at different temperatures, the collector discs retained Figure 2. 0.1%Tween
15min-Half
0.1%Tween
5min-Half
0.5%Tween
5min-Half
0.1%Tween
30min-Half
0.5%Tween
30min-Half
0.1%Tween
15min-
0.1%Tween
5min-
0.5%Tween
5min-
0.1%Tween
30min-
0.5%Tween
30min-storage at different temperatures, the collector discs retained
a collecting capacity of approximately 150 l of oral fluid per
g Day Day Day Day Day Overnight Overnight Overnight Overnight Overnight
a collecting capacity of approximately 150 l of oral fluid per
Escherichia coli cells (108 cells/ml) flushed with PBS at 1:1 (diluent:sample) ratio
Porex Treatment
disc The collector material costs are low and easy to build
Escherichia coli cells (108 cells/ml) flushed with PBS at 1:1 (diluent:sample) ratio Figure 3 – Average saliva absorbed by Porex pads under different treatmentsdisc. The collector material costs are low and easy to build
i t t ti h
Absorbance
Figure 3 Average saliva absorbed by Porex pads under different treatments
into any testing scheme.
Absorbance
it
Full saturation 60% saturationy g
capacity
%
Interaction Plot (data means) for Saliva absorbed (g)
Material
p y
305 overnighthalf-day
( ) (g)
2. Introduction Material
C V Sample recovered C V Sample recovered C V
305 overnighthalf day
Tween2. Introduction
g saliva/cm2 C.V.
( )
Sample recovered
( b b d)
C.V.
( )
Sample recovered
( b b d)
C.V.
( )
0.13
Tween
Conc(%)
g saliva/cm
(%) (% absorbed) (%) (% absorbed) (%) 0.1
0 5
Th f th ll t i t th d fi d
( ) ( ) ( ) ( ) ( ) Tween Conc(%) 0.12
0.5
The purpose of the collector is to gather a predefined Ultracell 1 2 5 2 84 1 0 78 2 6p p g p
oral specimen preserve the analytes and deliver an
Ultracell 1.2 5.2 84 1.0 78 2.6 0.11
oral specimen, preserve the analytes and deliver an PVA 1.2 0.9 69 3.8 68 1.9 0 13
Treatment
Time(min)
y
appropriate volume of specimen for future analysis
PVA 1.2 0.9 69 3.8 68 1.9
P 0 07 3 1 90 2 7
0.13 Time(min)
5
appropriate volume of specimen for future analysis. Porex 0.07 3.1 90 2.7 Treatment Time(min) 0.12
30
In order to accomplish this task the collector must
0.12
In order to accomplish this task, the collector must Escherichia coli cells (108 cells/ml) flushed with PBS at 3:1 (diluent:sample) ratio 0.11
have certain general features that include:
Escherichia coli cells (10 cells/ml) flushed with PBS at 3:1 (diluent:sample) ratio
have certain general features that include:
 A t h ld l ti bl th t An easy to hold plastic assembly that Ab b Drying Timeeasy to o d p ast c asse b y t at
i f t bl i th th
Absorbance
Full saturation 60% saturation
Drying Time
is comfortable in the mouth capacity
Full saturation 60% saturation
 The collector and assembly should be
capacity
 The collector and assembly should be Material
hygienic and easy to use
Material
li / 2 C.V. Sample recovered C.V. Sample recovered C.V. Figure 4 – Interaction plot for different treatment variables
hygienic and easy to use g saliva/cm2 C.V.
(%)
Sample recovered
(% b b d)
C.V.
(%)
Sample recovered
(% b b d)
C.V.
(%) A h dl d h ld bl f i f h h dl Materials on the collector must gather a
g
(%) (% absorbed) (%) (% absorbed) (%) A handle and a holder capable of separating from the handle were Materials on the collector must gather a A handle and a holder capable of separating from the handle were
d i d Th fi l d t i h i Fi 5predetermined volume of specimen Ult ll 1 2 4 7 89 1 7 86 0 8
designed. The final product is shown in Figure 5.predetermined volume of specimen
it bl f l i
Ultracell 1.2 4.7 89 1.7 86 0.8
g p g
suitable for analysis. PVA 1 2 1 9 80 2 7 77 2 4y
 The anal te m st be stable and
PVA 1.2 1.9 80 2.7 77 2.4
 The analyte must be stable and Porex 0 067 4 7 96 4 0y
recoverable from the collector
Porex 0.067 4.7 96 4.0
recoverable from the collector.
E h h l ll (103 ll / l) fl h d i h PBS 3 1 (dil l ) i The collector must engage to the assay Escherichia coli cells (103 cells/ml) flushed with PBS at 3:1 (diluent:sample) ratio The collector must engage to the assay ( ) ( p )
chip or storage receptacle and easilychip or storage receptacle and easily
deliver the specimen Absorbance
F ll i 60% i
deliver the specimen
S l ll ti t i l id d d
Absorbance
it
Full saturation 60% saturation
Several collecting materials were considered and capacityg
evaluated for their capacity to collect and release aevaluated for their capacity to collect and release a
Material
Figure 5 – Handle and holder designp y
biological sample for this a model was developed
Material
C V Sample recovered C V Sample recovered C V With th ll t d fi d l th d f l tbiological sample, for this a model was developed g saliva/cm2 C.V. Sample recovered C.V. Sample recovered C.V. With the collector defined, samples were gathered from volunteers
consisting of oral fluid samples spiked with bacterial
g saliva/cm
(%) (% absorbed) (%) (% absorbed) (%)
, p g
and used to measure the protein recovery and the volume collectedconsisting of oral fluid samples spiked with bacterial ( ) ( ) ( ) ( ) ( )
and used to measure the protein recovery and the volume collected
cells expressing green fluorescent protein (GFP)
p y
stability over an extended period The results of these studies arecells expressing green fluorescent protein (GFP).
GFP i i l d bi l i l k I i
stability over an extended period. The results of these studies are
GFP is routinely used as a biological marker. It is Ultracell 1.2 1.5 85 9 84 12.9 shown in Figures 6 and 7GFP is routinely used as a biological marker. It is
ti l l f l d t it t bilit d b i
shown in Figures 6 and 7.
particularly useful due to its stability and because is Porex 0.07 2.0 92 12p y y
easily detected exhibiting and excitation peak at 475
Porex 0.07 2.0 92 12
Total protein recovery from saliva samples absorbed in Porex pads
easily detected exhibiting and excitation peak at 475 Table 1 – Cell recovery after absorption and expression of sample through different collecting materials collectorsy g p
nm and emission at 508 nm
Table 1 – Cell recovery after absorption and expression of sample through different collecting materials.
nm and emission at 508 nm. 100%
Expression of E. coli cells through PVA, Ultracell sponges and 80%
(%)
3 Experimental method
p g , p g
Porex material
red
3.Experimental method Porex material
60%
over
100
reco
F th i t E h i hi li ll
40%
einr
For these experiments, Escherichia coli cells were 90
Prote
p ,
transformed with a plasmid containing the GFP open
d)
20%
P
transformed with a plasmid containing the GFP open 80
bed
p g p
reading frame (ORF) under the control of the
80
orb
0%
reading frame (ORF) under the control of the
bs
Subject 1 Subject 2 Subject 3
arabinose promoter The cells were subsequently
70
%a
arabinose promoter. The cells were subsequently
(%
Figure 6 – Total protein recovery from oral fluid samples gathered with collector
cultured and used to spike oral fluid samples which 60
ed
Figure 6 Total protein recovery from oral fluid samples gathered with collector
cultured and used to spike oral fluid samples which
b b d i t th t i l t t d d th
vere
Ult ll Stability study of Porex collection capacity,Stability study of Porex collection capacity,
were absorbed into the materials tested and then 50
cov
Ultracell Stability study of Porex collection capacity,
Subject 20 200
Stability study of Porex collection capacity,
Subject 1were absorbed into the materials tested and then
d Th ll t d b f b ti
50
rec
PVA 0.200
j0.200
j
expressed. The cells were counted before absorption
lsr
PVA
P 0 160
(g)
0.160
g)
p p
and after expression hence determining the number
40
cell
Porex 0.160
ed(
d(g
and after expression hence determining the number
lic
0.120
cte
Ambient Temp0.120
cted
Ambient Temp
g
of cells absorbed and released after expression 30
co
0 080
olle
37C
0 080
llec
Ambient Temp
37C
of cells absorbed and released after expression.
E
0.080
aco
Refrigerated
0.080
col
Refrigerated
Simple gravimetric measurements are used to 0.040
iva
0.040
va
Simple gravimetric measurements are used to 20
0 000
Sal
0.040
Saliv
determine amounts of sample collected (Figure 1)
0.000
0 14 28 40 56
S
0.000
S
determine amounts of sample collected (Figure 1) 10
Porex storage time (days)
0 14 28 40 56 70
Pore storage time (da s)
7
Porex storage time (days)Porex storage time (days)
1 2
7
8
0
Stability study of Porex collection capacity
4 5 61 2 4 5 6 8
0
100% saturation 60% saturation 100% saturation 60% saturation 100% saturation 60% saturation
Stability study of Porex collection capacity,
Subject 34 5 64 5 6 100% saturation 60% saturation 100% saturation 60% saturation 100% saturation 60% saturation
0 200
Subject 3
1:1 dilution 3:1 dilution 3:1 dilution
0.200
0 160
10^8 cells/ml 10^3 cells/ml
0.160
g)
0 120
ed(g
3
Figure 2– Cell recovery after absorption and expression of sample through different collecting materials.
0.120
ecte
3
coll
Ambient Temp
3 0.080
liva
37C
Refrigerated
D t th i di it b th PVA d Ult ll
Sal
Refrigerated
o Due to their expanding capacity, both PVA and Ultracell 0.040
p g p y,
sponges collect higher sample volumes than the rigid discsponges collect higher sample volumes than the rigid disc 0.000
0 14 28 40 56 70
4 E t 7 W i h S i
p g g p g
(Porex)
0 14 28 40 56 70
P t ti (d )
1 Weigh tube 4. Expose sponge to 7. Weigh Syringe (Porex). Porex storage time (days)
1. Weigh tube
f ll ti sample after expression o Similar recovery values were observed at both high and low Figure 6 – Sample volume stability test over 70 days at different storage temperatures
for collection sample
5 I t d
after expression
t d t i
o Similar recovery values were observed at both high and low Figure 6 – Sample volume stability test over 70 days at different storage temperatures
2 Weigh syringe 5. Introduce sponge to determine cell concentrations in the case of Ultracell and Porex 5 C l i2. Weigh syringe p g
in syringe and mass of sample
cell concentrations in the case of Ultracell and Porex.
A ll ll l i b d h h l
5. Conclusions
for expression in syringe and mass of sample o A small cell lysis rate was observed when the samples werefor expression
3 Weigh weigh to retained by
o A small cell lysis rate was observed when the samples were
th h Ult ll d P3. Weigh weigh to
d t i f
retained by
run through Ultracell and Porex.  Porex is a viable substrate for the collection of oral fluidscollecting determine mass of sponge.
g
As much as 20% of the cells are lysed when they pass through
 Porex is a viable substrate for the collection of oral fluids.collecting
sample collected 8 Weigh tube with o As much as 20% of the cells are lysed when they pass through  Analytes can be well preserved and recovered from the Porexsponge sample collected
6 Fl h ith
8. Weigh tube with
ll t d
y y p g
PVA
 Analytes can be well preserved and recovered from the Porexp g
6. Flush sponge with collected PVA. material if the surface tension is reducedp g
diluent sample o The Porex material exhibited the highest recovery of biological
material if the surface tension is reduced.
 Addi i l di d d d fi h li i i f Pdiluent sample o The Porex material exhibited the highest recovery of biological  Additional studies are needed to define the limitations of Porex
specimens with low cell lysing
Additional studies are needed to define the limitations of Porex
ith th l t
9
specimens with low cell lysing.
K i th t th P t i l i t i l t i
with other analytes.
9 9 Count cells and o Knowing that the Porex material is easy to manipulate in a
y
9. Count cells and o Knowing that the Porex material is easy to manipulate in a
f t i d th t it h b f ll d 6 Acknowledgments
calculate manufacturing process and that it has been successfully used 6. Acknowledgments
calculate
t ti
g p y
in the past in diagnostic applications we selected it as ourretention in the past in diagnostic applications, we selected it as our
NYU ll f d ti t P f D M l d D Willi
p g pp
material of choice to incorporate into the collector design NYU college of dentistry, Professor Dan Malamud, Dr. Williammaterial of choice to incorporate into the collector design. NYU college of dentistry, Professor Dan Malamud, Dr. William
Ab Ch l B b ll L hi h t d t Ni hit KAbrams, Cheryl Barber as well as Lehigh students Nishita K.Figure 1– Gravimetric procedure to determine volumes collected and recovered after expression , y g
Shastri William F S Peterson and Nickolas T Agathis
g p p
Shastri, William F S Peterson and Nickolas T. Agathisg