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OralDisk: A Chair-Side Compatible Molecular Platform Using Whole Saliva for Monitoring Oral Health at the Dental Practice

Authors:
  • Hahn-Schickard Freiburg
  • Hahn-Schickard, Freiburg

Abstract

Periodontitis and dental caries are two major bacterially induced, non-communicable diseases that cause the deterioration of oral health, with implications in patients' general health. Early, precise diagnosis and personalized monitoring are essential for the efficient prevention and management of these diseases. Here, we present a disk-shaped microfluidic platform (OralDisk) compatible with chair-side use that enables analysis of non-invasively collected whole saliva samples and molecular-based detection of ten bacteria: seven periodontitis-associated (Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Fusobacterium nucleatum, Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola) and three caries-associated (oral Lactobacilli, Streptococcus mutans, Streptococcus sobrinus). Each OralDisk test required 400 µL of homogenized whole saliva. The automated workflow included bacterial DNA extraction, purification and hydrolysis probe real-time PCR detection of the target pathogens. All reagents were pre-stored within the disk and sample-to-answer processing took < 3 h using a compact, customized processing device. A technical feasibility study (25 OralDisks) was conducted using samples from healthy, periodontitis and caries patients. The comparison of the OralDisk with a lab-based reference method revealed a ~90% agreement amongst targets detected as positive and negative. This shows the OralDisk's potential and suitability for inclusion in larger prospective implementation studies in dental care settings.
biosensors
Article
OralDisk: A Chair-Side Compatible Molecular Platform Using
Whole Saliva for Monitoring Oral Health at the Dental Practice
Desirée Baumgartner 1, 2, *, Benita Johannsen 1, Mara Specht 1, Jan Lüddecke 1, Markus Rombach 1, Sebastian Hin 1,
Nils Paust 1,2, Felix von Stetten 1,2 , Roland Zengerle 1,2, Christopher Herz 3, Johannes R. Peham 3,
Pune N. Paqué4, Thomas Attin 4, Joël S. Jenzer 4, Philipp Körner 4, Patrick R. Schmidlin 4,
Thomas Thurnheer 4, Florian J. Wegehaupt 4, Wendy E. Kaman 5,6, Andrew Stubbs 7, John P. Hays 5,
Viorel Rusu 8, Alex Michie 9, Thomas Binsl 9, David Stejskal 10,11 , Michal Karpíšek 12, 13 , Kai Bao 14,
Nagihan Bostanci 14, Georgios N. Belibasakis 14 and Konstantinos Mitsakakis 1 ,2 ,*


Citation: Baumgartner, D.;
Johannsen, B.; Specht, M.; Lüddecke,
J.; Rombach, M.; Hin, S.; Paust, N.;
von Stetten, F.; Zengerle, R.; Herz, C.;
et al. OralDisk: A Chair-Side
Compatible Molecular Platform
Using Whole Saliva for Monitoring
Oral Health at the Dental Practice.
Biosensors 2021,11, 423. https://
doi.org/10.3390/bios11110423
Received: 1 October 2021
Accepted: 24 October 2021
Published: 28 October 2021
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Copyright: © 2021 by the authors.
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This article is an open access article
distributed under the terms and
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Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany;
Benita.Johannsen@Hahn-Schickard.de (B.J.); Mara.Specht@Hahn-Schickard.de (M.S.);
Jan.Lueddecke@Hahn-Schickard.de (J.L.); Markus.Rombach@Hahn-Schickard.de (M.R.);
sebastian.hin@iuvas.de (S.H.); Nils.Paust@Hahn-Schickard.de (N.P.);
Felix.von.Stetten@Hahn-Schickard.de (F.v.S.); Roland.Zengerle@Hahn-Schickard.de (R.Z.)
2Laboratory for MEMS Applications, IMTEK–Department of Microsystems Engineering,
University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany
3AIT Austrian Institute of Technology, Molecular Diagnostics, Giefinggasse 4, 1210 Wien, Austria;
christopher_herz@pall.com (C.H.); Johannes.Peham@ait.ac.at (J.R.P.)
4Clinic of Conservative and Preventive Dentistry, Center of Dental Medicine, University of Zurich,
Plattenstrasse 11, 8032 Zurich, Switzerland; punenina.paque@zzm.uzh.ch (P.N.P.);
thomas.attin@zzm.uzh.ch (T.A.); Joel.Jenzer@icloud.com (J.S.J.); philipp.koerner@zzm.uzh.ch (P.K.);
patrick.schmidlin@zzm.uzh.ch (P.R.S.); Thomas.Thurnheer@zzm.uzh.ch (T.T.);
florian.wegehaupt@zzm.uzh.ch (F.J.W.)
5Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Centre
Rotterdam (Erasmus MC), 3015 CN Rotterdam, The Netherlands; w.e.kaman@acta.nl (W.E.K.);
j.hays@erasmusmc.nl (J.P.H.)
6Department of Oral Biochemistry, Academic Centre for Dentistry Amsterdam (ACTA), Free University of
Amsterdam and University of Amsterdam, 1081 LA Amsterdam, The Netherlands
7Department of Pathology and Clinical Bioinformatics, Erasmus University Medical Centre
Rotterdam (Erasmus MC), 3015 CN Rotterdam, The Netherlands; a.stubbs@erasmusmc.nl
8Magtivio B.V., Daelderweg 9, 6361 HK Nuth, The Netherlands; vru@magtivio.com
9ClinicaGeno Ltd., 11 Station Approach, Coulsdon CR5 2NR, UK; alex@clinicageno.com (A.M.);
thomas@clinicageno.com (T.B.)
10 Department of Biomedical Sciences, Faculty of Medicine, University of Ostrava, Syllabova 19,
70300 Ostrava, Czech Republic; david.stejskal@fno.cz
11 Institute of Laboratory Diagnostics, University Hospital Ostrava, 17. Listopadu 1790/5,
70800 Ostrava, Czech Republic
12
BioVendor-LaboratorníMedicína a.s., Research & Diagnostic Products Division, Karasek 1767/1, Reckovice,
62100 Brno, Czech Republic; karpisek@biovendor.com
13 Faculty of Pharmacy, Masaryk University, Palackeho trida 1946/1, 61242 Brno, Czech Republic
14 Section of Oral Health and Periodontology, Division of Oral Diseases, Department of Dental Medicine,
Karolinska Institutet, 14104 Huddinge, Sweden; kai.bao@ki.se (K.B.); nagihan.bostanci@ki.se (N.B.);
george.belibasakis@ki.se (G.N.B.)
*Correspondence: Konstantinos.Mitsakakis@Hahn-Schickard.de (K.M.);
Desiree.Baumgartner@Hahn-Schickard.de (D.B.); Tel.: +49-761-203-73252 (K.M.); +49-761-203-98724 (D.B.)
Abstract:
Periodontitis and dental caries are two major bacterially induced, non-communicable dis-
eases that cause the deterioration of oral health, with implications in patients’ general health. Early, pre-
cise diagnosis and personalized monitoring are essential for the efficient prevention and management
of these diseases. Here, we present a disk-shaped microfluidic platform (OralDisk) compatible with
chair-side use that enables analysis of non-invasively collected whole saliva samples and molecular-
based detection of ten bacteria: seven periodontitis-associated (
Aggregatibacter actinomycetemcomitans
,
Campylobacter rectus
,
Fusobacterium nucleatum
,Prevotella intermedia,Porphyromonas gingivalis,
Tannerella forsythia,Treponema denticola) and three caries-associated (oral Lactobacilli,Streptococcus mutans
,
Streptococcus sobrinus). Each OralDisk test required 400
µ
L of homogenized whole saliva. The
Biosensors 2021,11, 423. https://doi.org/10.3390/bios11110423 https://www.mdpi.com/journal/biosensors
Biosensors 2021,11, 423 2 of 21
automated workflow included bacterial DNA extraction, purification and hydrolysis probe real-time
PCR detection of the target pathogens. All reagents were pre-stored within the disk and sample-to-
answer processing took < 3 h using a compact, customized processing device. A technical feasibility
study (25 OralDisks) was conducted using samples from healthy, periodontitis and caries patients.
The comparison of the OralDisk with a lab-based reference method revealed a ~90% agreement
amongst targets detected as positive and negative. This shows the OralDisk’s potential and suitability
for inclusion in larger prospective implementation studies in dental care settings.
Keywords:
dental practice; point-of-care diagnostics; treatment monitoring; oral health; periodontitis;
caries; saliva diagnostics
1. Introduction
Oral diseases are the most prevalent chronic diseases worldwide, accounting for
almost 5 billion cases globally [
1
]. They are the third most expensive group of diseases
to treat in the EU, following diabetes and cardiovascular diseases [
2
]. In addition, the
overprescription of antibiotics in dentistry is a challenge [
3
]. Indicatively, up to 80% of
prophylactic antibiotic use in the US is considered unjustified [4].
The two most prevalent oral microbial diseases are caries and periodontitis. Dental
caries affects the hard tissue of the teeth, causing tooth decay. Periodontitis affects the
tissues that surround and support the teeth, leading to progressive loss of the bone and
soft tissue attachment and eventually tooth loss. There are different clinical manifestations
and degrees of severity of periodontal disease. According to the Global Burden of Disease
(GBD) study, 796 million people around the globe had severe periodontitis in 2017, ranking
it within the top 10 most prevalent conditions worldwide [
5
]. Across Europe, 5–20% of
middle-aged people and up to 40% of elderly people are affected by it [
6
]. In the US, 80% of
the population has some form of periodontal disease [
7
]. Periodontitis has also been related
to systemic diseases such as type 2 diabetes mellitus (T2DM), cardiovascular diseases,
Alzheimer’s disease and others [
8
10
]. Therefore, being able to detect periodontitis-causing
bacteria has a much broader clinical significance than simply monitoring oral health, as it
can potentially provide a signal for deteriorating systemic health [11].
Caries and periodontitis are both treatable, and the rationale around their treatment
is the same, namely, the removal of the microbial biofilm which is the causative factor.
The treatment of periodontal disease, upon the removal of the biofilm, requires good oral
hygiene protocols and monitoring to ensure that the inflammation has subsided. For dental
caries, upon removal of the infective caries tissue, the treatment mandates restoration of
the lost hard tissue with appropriate restorative material (fillings). Although the treatment
protocols for both diseases are well-defined, there exist urgent and unmet medical (dental)
needs related to: (i) early diagnosis (even before symptoms emerge), which would assist
in preventing the disease, benefiting the patients’ quality of life while also saving on the
costs of treatment and (ii) accurate monitoring during and after treatment (i.e., during
the maintenance phase of treatment) in cases where a patient presents with advanced
or aggressive disease. Accurate monitoring would allow the dentist to make the correct
assessment of when to start and finish treatment: not too early, running the risk of re-
emergence, and not too late, thus spending unnecessary resources.
The current state of the art for performing such diagnoses and monitoring is still
largely dependent on clinical examination, patient history and radiographic imaging
(
X-rays
). However, radiographs mainly observe damage that occurred in the past, while
plain clinical examinations tend to miss the first signs of incipient dysbiosis and therefore
do not contribute to early detection and prevention. Furthermore, cumulative X-ray ra-
diation during periodontal treatment and frequent visits for follow-up monitoring poses
health risks and special concerns in some patients (e.g., pregnant women). Periodontal
probing is a frequently used methodology in which a probe is inserted into the gingival
Biosensors 2021,11, 423 3 of 21
sulcus in order to measure pocket depths around a tooth and to assess the health status of
the periodontium [
12
,
13
]. However, it is an invasive method in the gums, and as such, it
should not be used in patients with T2DM as it may lead to bacteremia [
14
,
15
], thereby in-
creasing systemic inflammation and the infection risk in already vulnerable T2DM patients.
Additionally, general clinical examinations do not include probing of the whole gingival
sulcus of the teeth. Other typical (simplified) measurements for basic periodontal screening
include the community periodontal index (CPI) and the basic periodontal examination
(BPE) [
16
], which are performed by looking for increased pocket depths and bleeding
on probing.
Unfortunately, these current ’gold-standard’ diagnostic approaches may risk missing
the diagnosis of emerging periodontitis. Therefore, complementary diagnostic tools, which
are largely less invasive and less tedious, could improve the diagnostic sensitivity, precision
and accuracy, for example, by screening the microbial ecology of the oral cavity in order to
help indicate whether a patient needs further diagnostic evaluation and treatment or to
monitor patients’ post-treatment status.
In this respect, the current publication proposes a rapid, molecular-based and non-
invasive platform for detecting oral disease-causing bacteria, with a workflow that is
compatible with point-of-care (POC) dental settings [
17
] and which can function as an
auxiliary tool to the current gold standard diagnostic methods. The OralDisk microfluidic
cartridge integrates all the biochemical reagents needed for fully automated analysis,
namely: (i) customized buffers and microfluidic-optimized magnetic particles for the
purification of bacterial DNA; (ii) amplification reagents in a lyophilized form; and (iii) POC-
ready real-time qPCR TaqMan primers/probes for specific bacterial detection. The OralDisk
is an application-specific version of the centrifugal microfluidic LabDisk platform, which
has already demonstrated its utility in applications where a single infectious pathogen
is to be detected [
18
21
]. This small-scale technical feasibility study demonstrates for
the first time the platform’s implementation in the field of oral health, where multiple
bacteria may be present simultaneously in the oral cavity and whole saliva is used as
diagnostic specimen. It presents data on the detection of three caries-associated and
seven periodontitis-associated bacterial species in complex saliva samples collected from
individuals that were classified into three study groups (healthy, caries and periodontitis)
following an assessment by dental specialists. The patients’ status assessment and the
sample collection were performed in a previous clinical study [
22
]. The results from the
OralDisk were compared with a lab-based extraction and qPCR reference method, as
well as with the iai PadoTest (Institut für Angewandte Immunologie IAI AG, Zuchwil,
Switzerland) commercial reference method. The OralDisk exhibited comparable (and in
some cases superior) behavior while offering the additional benefit of automation.
2. Materials and Methods
2.1. Sample Collection and Ethics Permission
The samples used in this study were a sub-group (n= 24) of a large cohort (n= 214) of
samples that had been collected at the Center for Dental Medicine, University of Zurich.
These samples were intended for the microbial analysis of saliva with the aim of identifying
oral infections in patients [
22
]. The 24 samples (seven healthy, nine caries and eight
periodontitis samples) were selected in order to demonstrate the technical feasibility of the
OralDisk, without the intention of generating clinical conclusions. The sample collection
and the study protocol were approved by the local Swiss ethics committee (BASEC-no.
2016-00435) and all sample donors signed a written informed consent form prior to saliva
collection. The collection contained unstimulated whole saliva that was aliquoted and
stored at
80
C until further analysis. Details on the inclusion/exclusion criteria and on
the collection methodology are available in Paquéet al. [22].
Biosensors 2021,11, 423 4 of 21
2.2. Selected Bacterial Panel
Quantitative shifts in the levels of multiple, rather than single, bacterial species may
more accurately reflect dysbiotic changes in oral microbial ecology that are commensu-
rate with the initiation or progression of oral disease [
23
]. Measurement of the species
dynamics of oral polymicrobial populations is the key element that allows potential moni-
toring for the prevention, early diagnosis and post-treatment follow-up of oral diseases.
Therefore, ten bacteria were included in the OralDisk panel: seven Gram-negative bac-
teria related to periodontitis (Aggregatibacter actinomycetemcomitans,Campylobacter rectus,
Fusobacterium nucleatum
,Prevotella intermedia,Porphyromonas gingivalis,Tannerella forsythia
and Treponema denticola) and three Gram-positive bacteria associated with caries (oral
associated Lactobacilli,Streptococcus mutans and Streptococcus sobrinus).
This panel was chosen based on: (i) current knowledge on the association of certain
oral bacteria with caries and periodontitis [
24
]; (ii) feedback from experts in response to sur-
vey questionnaires; and (iii) the goal of including bacteria which were identified in subgin-
gival [
25
] and supragingival [
26
] biofilm samples of subjects with and without periodontitis.
Furthermore, the role of these bacteria as differentiators between healthy, periodontitis and
caries groups was demonstrated in a preceding clinical study by
Paquéet al.
[
22
], which
provided additional evidence for including these particular bacteria in the current technical
feasibility study of the OralDisk. In the aforementioned clinical study, statistically signif-
icant polymicrobial differentiators were observed (i) between healthy and periodontitis
groups (C. rectus,T. forsythia,P. gingivalis,S. mutans,F. nucleatum,T. denticola,P. intermedia
and oral Lactobacilli); (ii) between healthy and caries groups (S. mutans and T. denticola);
and (iii) between caries and periodontitis groups (S. mutans).
2.3. Reference Method #1: Lab-Based DNA Extraction and qPCR
Enzymatic lysis was performed on 920
µ
L of whole saliva using the GenElute
TM
Bacterial Genomic DNA Kit (Sigma-Aldrich, Saint Louis, MO, USA), followed by silica
column-based extraction (Figure 1). An adjusted manufacturer’s protocol was used, as
described in previous work [
22
]. An eluate volume of 135
µ
L was stored at
25
C and later
used to perform qPCR (Roche LightCycler). Details on the POC-compatible qPCR assay
development, primer/probe design, assay validation (including qPCR assay sensitivity
and limit of detection), utilized amplification conditions and data analysis, are available in
Paquéet al. [22].
Biosensors 2021, 11, x FOR PEER REVIEW
5 of 22
Figure 1. Experimental workflows from sample collection until analysis.
2.4. Reference Method #2: Commercial iai PadoTest
The iai PadoTest (Institut für Angewandte Immunologie IAI AG, Zuchwil,
Switzerland) [27] is a commercially available test that was used as a second reference to
the OralDisk (Figure 1). It performs a multiplex real-time qPCR assay that estimates
bacterial cell counts based on 16S rRNA [28]. The manufacturer’s collection protocol
stipulates that paper points are inserted into dental pockets to collect gingival crevicular
fluid (GCF). The paper points are stored in vials (one or more paper points per vial are
possible, in single- or pool-mode of analysis). To make the results comparable with our
saliva-based detection, the sample collection protocol was slightly modified: four paper
points (Roeko Iso 55, Coltène, Altstätten, Switzerland) were immersed in 40 µL of thawed
whole saliva in a tube. The tubes with the paper points were sent to iai PadoTest AG for
analysis.
2.5. Mechanical Lysis and Homogenization of Saliva Samples Prior to Insertion into the
OralDisk
Lysis of bacteria, with simultaneous homogenization of the saliva, was performed
prior to insertion into the OralDisk by means of a mechanical bead-beating process using
a hand-held device (Terralyzer, Zymo Research, Irvine, CA, USA). Notably, this was the
only manual step in the protocol (Figure 1). A volume of 600 µL of whole saliva and 10
µL of 1:15 (or 1:10 for two samples) diluted Gram-positive bacterium Serinicoccus marinus
[29] (process control [22]) was inserted into a 2-mL tube with 1.30 g of 0.2-mm steel beads
(Next Advance Inc., Troy, NY, USA). The tube was then inserted into the Terralyzer. The
bead-beating protocol for all samples was 2 × 10 s with a 20 s break (apart from samples
GTT33 and KxTC22, for which it was 2 × 20 s with a 10 s break).
2.6. OralDisk Design and Workflow for Fully Automated Real-Time PCR
Oral bacteria were detected using the centrifugal microfluidic OralDisk, which
incorporated all the microfluidic unit operations [30] required for the fully automated
analysis of whole saliva samples. A volume of 400 µL of ex situ homogenized saliva
(Section 2.5) was inserted into the OralDisk (Figure 2, #1) and on-disk DNA extraction and
purification was based on a bind-wash-elute protocol [31]. Dedicated buffers were
developed by magtivio B.V., the Netherlands, and were stored in pouches (stickpacks
[32]) on the disk (Figure 2, #2a–2d). Upon centrifugation (and assisted by controlled
heating), liquids were released into their respective (radially outward) chambers (Figure
Collected whole saliva
Aliquot #1
(920 µL)
Aliquot #2
(40 µL)
Aliquot #3
(600 µL)
Bead-beating 610 µL
(with Terralyzer)
Insert 400 µL into OralDisk
(automated extraction and
PCR)
Addition of
10 µL control bacterium
S. marinus
4 paper points immersed
in saliva
Real-time qPCR from paper
points @ iai PadoTest
Enzymatic lysis
Column purification
0.01 ng S. marinus genomic
DNA spiked in PCR tube
Real-time qPCR on
benchtop equipment
Lab-based reference Commercial reference OralDisk
Figure 1. Experimental workflows from sample collection until analysis.
Biosensors 2021,11, 423 5 of 21
2.4. Reference Method #2: Commercial iai PadoTest
The iai PadoTest (Institut für Angewandte Immunologie IAI AG, Zuchwil, Switzer-
land) [
27
] is a commercially available test that was used as a second reference to the
OralDisk (Figure 1). It performs a multiplex real-time qPCR assay that estimates bacterial
cell counts based on 16S rRNA [
28
]. The manufacturer’s collection protocol stipulates that
paper points are inserted into dental pockets to collect gingival crevicular fluid (GCF). The
paper points are stored in vials (one or more paper points per vial are possible, in single- or
pool-mode of analysis). To make the results comparable with our saliva-based detection,
the sample collection protocol was slightly modified: four paper points (Roeko Iso 55,
Coltène, Altstätten, Switzerland) were immersed in 40
µ
L of thawed whole saliva in a tube.
The tubes with the paper points were sent to iai PadoTest AG for analysis.
2.5. Mechanical Lysis and Homogenization of Saliva Samples Prior to Insertion into the OralDisk
Lysis of bacteria, with simultaneous homogenization of the saliva, was performed
prior to insertion into the OralDisk by means of a mechanical bead-beating process using a
hand-held device (Terralyzer, Zymo Research, Irvine, CA, USA). Notably, this was the only
manual step in the protocol (Figure 1). A volume of 600
µ
L of whole saliva and 10
µ
L of
1:15 (or 1:10 for two samples) diluted Gram-positive bacterium Serinicoccus marinus [
29
]
(process control [
22
]) was inserted into a 2-mL tube with 1.30 g of 0.2-mm steel beads
(Next Advance Inc., Troy, NY, USA). The tube was then inserted into the Terralyzer. The
bead-beating protocol for all samples was 2
×
10 s with a 20 s break (apart from samples
GTT33 and KxTC22, for which it was 2 ×20 s with a 10 s break).
2.6. OralDisk Design and Workflow for Fully Automated Real-Time PCR
Oral bacteria were detected using the centrifugal microfluidic OralDisk, which incor-
porated all the microfluidic unit operations [
30
] required for the fully automated analysis
of whole saliva samples. A volume of 400
µ
L of ex situ homogenized saliva (Section 2.5)
was inserted into the OralDisk (Figure 2, #1) and on-disk DNA extraction and purifica-
tion was based on a bind-wash-elute protocol [
31
]. Dedicated buffers were developed
by magtivio B.V., the Netherlands, and were stored in pouches (stickpacks [
32
]) on the
disk (Figure 2, #2a–2d). Upon centrifugation (and assisted by controlled heating), liquids
were released into their respective (radially outward) chambers (Figure 2, #4a–4d). The
stickpacks contained 440
µ
L of binding buffer (#2a); 200
µ
L of wash buffer 1 (#2b); 200
µ
L
of wash buffer 2 (#2c); and 180
µ
L of elution buffer (#2d). Magnetic beads (MagSi-DNA
mf beads, ferrimagnetic core with silica shell, cat. no. MD0200010002) were developed
by magtivio B.V. especially for microfluidic use for this application and were dry-stored
on the disk (
Figure 2
, #3). Upon magnetic bead rehydration by the binding buffer and
lysate, the magnetic beads captured the DNA and were transported through the sub-
sequent chambers (Figure 2, #4a–4d) by means of controlled continuous disk rotation
and integrated magnets [
33
]. In chamber #4d (Figure 2), the DNA was eluted from the
magnetic beads, and 160
µ
L of the eluate was pumped radially inwards into chamber #6,
through structure #5 and by means of temperature change-rate (TCR) actuated valving [
34
]
and centrifugo-dynamic inward pumping [
35
]. In chamber #6, the amplification reagents
were pre-stored in the form of a lyophilized pellet (46
µ
L; TaqMan
®
Lyophilized 1-Step
qPCR Master Mix; 3.5
×
, Thermo Fisher Scientific, USA). Upon lyopellet rehydration and
thorough mixing using a dedicated microfluidic protocol to ensure homogeneity [
36
], the
mixture was aliquoted [
37
] into the PCR reaction chambers (#7) where the primers/probes
for each oral bacterium (plus those for the control bacterium S. marinus) were dry-stored.
Chamber (i) was a sacrificial chamber to collect residual liquid. Upon rehydration, the
thermocycling protocol for real-time PCR started: 95
C for 3 min (initial denaturation) and
40 cycles of 95 C for 10 s and 60 C for 30 s.
Biosensors 2021,11, 423 6 of 21
Biosensors 2021, 11, x FOR PEER REVIEW
7 of 22
Figure 2. OralDisk design. Blue sector: magnetic bead-based extraction and purification of DNA. #1:
sample inlet; #2: stickpacks for storage of buffers for: binding (2a), 1st washing (2b), 2nd washing
(2c) and elution (2d); #3: pre-stored (air-dried) magnetic beads; #4: chambers for binding (4a),
washing (4b, 4c) and elution of DNA from magnetic beads (4d). Grey sector: eluate transfer module,
automating the inward pumping (#5) and eluate mixing with the lyopellet (#6). Red sector:
amplification module, automating the preparation and execution of the real-time PCR in structure #7
in the reaction chambers labelled as (i)–(xiii). Structure #8 assists the liquid transfer from chamber #6
to the PCR structure #7.
Figure 3. Image of the items which comprise the experimental setup. (1) Tube containing the mixture
of the saliva sample, S. marinus control bacterium and steel beads. (2) Hand-held device (Terralyzer),
into which the tube is inserted for performing the mechanical lysis and saliva homogenization. (3)
Figure 2.
OralDisk design. Blue sector: magnetic bead-based extraction and purification of DNA.
#1: sample inlet; #2: stickpacks for storage of buffers for: binding (2a), 1st washing (2b), 2nd
washing (2c) and elution (2d); #3: pre-stored (air-dried) magnetic beads; #4: chambers for binding
(4a), washing (4b, 4c) and elution of DNA from magnetic beads (4d). Grey sector: eluate transfer
module, automating the inward pumping (#5) and eluate mixing with the lyopellet (#6). Red sector:
amplification module, automating the preparation and execution of the real-time PCR in structure #7
in the reaction chambers labelled as (i)–(xiii). Structure #8 assists the liquid transfer from chamber #6
to the PCR structure #7.
The microfluidic protocol comprised a slightly modified version of a protocol previ-
ously published by the authors [
21
]. According to this slightly modified protocol, the times
lapsed during the microfluidic processes in the blue-, grey- and red-marked modules in
Figure 2were: ~37 min, ~12 min and ~107 min, respectively (the latter including the PCR
thermocycling). Notably, the OralDisk does not have any sample outlet port but is a closed
system, as are all microfluidic cartridges in similar systems, so that the amplified DNA
does not contaminate the cartridge processing instrument, thereby risking false positive
results during the next measurement. Since we used human sample material, the disposal
of the OralDisk was performed by autoclaving, as for other typical laboratory consumables
(e.g., wells, tubes) that are used in nucleic acid amplification practices.
PCR thermocycling was performed in a customized LabDisk processing device func-
tional model (QIAGEN Lake Constance, currently DIALUNOX GmbH, Lake Constance,
Germany) (Figure 3) comprising: (i) a thermal module that enables global air heating for
performing the necessary thermocycling protocols; (ii) a mechanical module for the precise
positioning, acceleration and deceleration of the disks; (iii) an optical module for detection
of the real-time fluorescence signal derived from the nucleic acid amplification product; and
(iv) integrated magnets for bead transfer during the DNA extraction and purification. The
raw data acquired with the LabDisk Player were analyzed using a RotorGene (QIAGEN,
Hilden, Germany) software program to acquire Cq values.
2.7. OralDisk Fabrication
The OralDisks were fabricated by microthermoforming [
38
,
39
] of polycarbonate (PC)
polymer foils (250
µ
m thickness, Makrofol
®
DE 1-1 000000, Covestro, Leverkusen, Ger-
many) using a hot embossing machine (HEX01, Jenoptik AG, Jena, Germany) at the
Hahn-Schickard Lab-on-a-Chip Foundry Service [
40
]. Microthermoforming technology
is well-known from macro-scale blister package fabrication, which has been adapted and
Biosensors 2021,11, 423 7 of 21
transferred to the micro-scale. In short, an elastomeric mold made of poly(dimethylsiloxane)
(PDMS) was heated. The overlying polymer foil was heated as well, and at a specific tem-
perature above the foil’s glass transition, air was blown onto it so that it assumed the shape
of the mold. Ultimately, this technology possesses the following advantages: (i) it allows
monolithic fabrication of the cartridge and (ii) it is scalable and able to produce several
tens of thousands of pieces when required. After the foil structuring, a Teflon coating
(0.5% w/wTeflon (Teflon Amorphous Fluoropolymer, Chemours International Operations
Sarl, Geneva, Switzerland) in Fluorinert
FC-770 (art. # F3556-100ML, Sigma-Aldrich
Chemie GmbH, Darmstadt, Germany)) was applied to chambers #4a–4d (Figure 2) in
order to provide hydrophobic microfluidic properties in the nucleic acid extraction module.
Then, 20
µ
L of magnetic beads with 10
µ
L of 250 mg/mL trehalose were pipetted into
the corresponding chamber (Figure 2, #3), and 3.5
µ
L of each primer/probe reaction mix
with 0.5
µ
L of 1 M trehalose (final concentration 50 mM) were pipetted into each reaction
chamber (Figure 2, #7). The Teflon coating and the drying of the magnetic beads and
primers/probes followed a previously published protocol (1 h at 50
C) [
20
,
41
,
42
]. The
lyopellets containing the amplification reagents were manually inserted into the disk. All
buffers were stored in dedicated aluminum pouches (stickpacks). The sealing temperature
and pressure used to prepare the stickpacks allowed their opening at a specific rotational
frequency (70 Hz), whereby buffers were released into the corresponding chambers of
the extraction module (Figure 2). The stickpacks were also manually inserted into the
disk. The whole cartridge was sealed using a pressure-sensitive adhesive foil (9795R, 3M,
Maplewood, MN, USA). The cartridge was then inserted into an aluminum pouch with a
nitrogen atmosphere and desiccant bags, and was stored at room temperature until use.
Biosensors 2021, 11, x FOR PEER REVIEW
7 of 22
Figure 2. OralDisk design. Blue sector: magnetic bead-based extraction and purification of DNA. #1:
sample inlet; #2: stickpacks for storage of buffers for: binding (2a), 1st washing (2b), 2nd washing
(2c) and elution (2d); #3: pre-stored (air-dried) magnetic beads; #4: chambers for binding (4a),
washing (4b, 4c) and elution of DNA from magnetic beads (4d). Grey sector: eluate transfer module,
automating the inward pumping (#5) and eluate mixing with the lyopellet (#6). Red sector:
amplification module, automating the preparation and execution of the real-time PCR in structure #7
in the reaction chambers labelled as (i)–(xiii). Structure #8 assists the liquid transfer from chamber #6
to the PCR structure #7.
Figure 3. Image of the items which comprise the experimental setup. (1) Tube containing the mixture
of the saliva sample, S. marinus control bacterium and steel beads. (2) Hand-held device (Terralyzer),
into which the tube is inserted for performing the mechanical lysis and saliva homogenization. (3)
Figure 3.
Image of the items which comprise the experimental setup. (
1
) Tube containing the mixture
of the saliva sample, S. marinus control bacterium and steel beads. (
2
) Hand-held device (Terralyzer),
into which the tube is inserted for performing the mechanical lysis and saliva homogenization.
(
3
) The OralDisk, where the lysate is pipetted in the chamber indicated by the two white arrows.
(4) The LabDisk Player instrument that performs the OralDisk processing and real-time PCR.
Biosensors 2021,11, 423 8 of 21
2.8. Statistics
The p-values for T. forsythia were calculated using the pairwise Wilcoxon Rank Sum
Test (statistical software R [
43
] including the package tidyverse [
44
]) without correction for
multiple testing and with a significance level of 0.05. p-values were also calculated for
P. gingivalis
but were higher than 0.05 with both the OralDisk and the lab-based reference
method. For these two bacteria, all Cq measurements were available (i.e., no ‘ND’ values
in Supplementary Table S1). For the other eight bacterial species, one or more Cq values
were missing and were deemed to be beyond the limit of detection (marked as ‘ND’ in
Supplementary Table S1). These datasets were not used for the calculation of p-values. For
all species and diagnosis groups, we generated boxplots (Supplementary Figure S1) using
Origin
®
2019 software (version 2019 (9.60)) and included a calculation of the median values
and interquartile ranges (IQR) of the Cq results (Supplementary Table S2).
3. Results and Discussion
3.1. Real-Time PCR on the OralDisk
In this study, 25 disks were used to test 24 clinical samples (from seven healthy, nine
caries and eight periodontitis patients) and one negative control (H
2
O). Each sample was
tested once per disk. Figure 4shows representative OralDisk real-time PCR curves from
which the Cq values were calculated for all bacteria that were detected in a single sample.
The Cq values of all bacteria in all the samples tested with OralDisks are summarized
in Supplementary Table S1. These values were used for the subsequent analysis and
the comparison with the lab-based reference and commercial iai PadoTest methods. The
methods were compared by means of: (i) the number of assay targets detected as positive
and negative by the OralDisk, the iai PadoTest and the lab-based reference method and
(ii) the scatter plots of the acquired Cq values for each clinical diagnosis group and for
each bacterium for the OralDisk and the lab-based reference method. These analyses were
performed in order to examine possible trends in the data.
Biosensors 2021, 11, x FOR PEER REVIEW
9 of 22
which the Cq values were calculated for all bacteria that were detected in a single sample.
The Cq values of all bacteria in all the samples tested with OralDisks are summarized in
Supplementary Table S1. These values were used for the subsequent analysis and the
comparison with the lab-based reference and commercial iai PadoTest methods. The
methods were compared by means of: (i) the number of assay targets detected as positive
and negative by the OralDisk, the iai PadoTest and the lab-based reference method and
(ii) the scatter plots of the acquired Cq values for each clinical diagnosis group and for
each bacterium for the OralDisk and the lab-based reference method. These analyses were
performed in order to examine possible trends in the data.
Figure 4. Representative real-time PCR curves for the oral bacteria detected with the OralDisk in
one whole saliva sample.
3.2. Performance Comparison between the OralDisk and the Lab-Based Reference Method
Each saliva sample was tested using one OralDisk, which simultaneously screened
for ten bacterial species by means of its geometric multiplexing configuration. None of the
samples tested were found to contain all ten of the bacterial species screened for. This was
in line with our previous findings using a full cohort study, in which the corresponding
lab-based PCR reference method was used [22]. In order to assess the degree of qualitative
agreement (i.e., bacterial presence/absence) between the OralDisk and the corresponding
lab-based reference method, we divided the results (Table 1) into the following four
groups:
(a) assay targets detected as positive by both the OralDisk and the lab-based reference
(agreement in positive samples: 154/175 (88.0%) cases);
(b) assay targets detected as positive by the OralDisk but negative by the lab-based
reference (disagreement in 7/59 (11.9%) cases);
(c) assay targets detected as negative by both the OralDisk and the lab-based reference
(agreement in negative samples: 52/59 (88.1%) cases);
(d) assay targets detected as negative by the OralDisk but positive by the lab-based
reference (disagreement in 21/175 (12.0%) cases).
-0.05
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0 5 10 15 20 25 30 35 40
Normalized Fluorescence
Cq OralDisk
OralDisk real-time PCR curves from a representative sample
C. rectus
F. nucleatum
P. intermedia
P. gingivalis
T. forsythia
T. denticola
S. mutans
S. sobrinus
Figure 4.
Representative real-time PCR curves for the oral bacteria detected with the OralDisk in one
whole saliva sample.
3.2. Performance Comparison between the OralDisk and the Lab-Based Reference Method
Each saliva sample was tested using one OralDisk, which simultaneously screened for
ten bacterial species by means of its geometric multiplexing configuration. None of the
samples tested were found to contain all ten of the bacterial species screened for. This was
Biosensors 2021,11, 423 9 of 21
in line with our previous findings using a full cohort study, in which the corresponding
lab-based PCR reference method was used [
22
]. In order to assess the degree of qualitative
agreement (i.e., bacterial presence/absence) between the OralDisk and the corresponding
lab-based reference method, we divided the results (Table 1) into the following four groups:
(a)
assay targets detected as positive by both the OralDisk and the lab-based reference
(agreement in positive samples: 154/175 (88.0%) cases);
(b)
assay targets detected as positive by the OralDisk but negative by the lab-based
reference (disagreement in 7/59 (11.9%) cases);
(c)
assay targets detected as negative by both the OralDisk and the lab-based reference
(agreement in negative samples: 52/59 (88.1%) cases);
(d)
assay targets detected as negative by the OralDisk but positive by the lab-based
reference (disagreement in 21/175 (12.0%) cases).
A comparison between the OralDisk and the lab-based reference results was per-
formed (i) for each clinical diagnosis group and (ii) for each bacterial species. Regarding
the former, the highest agreement between the two methods in terms of positively detected
targets (group (a)) was found in the caries samples (91.9%), followed by the healthy samples
(85.4%) and, finally, the periodontitis samples (85.0%). Regarding the latter, the positive
agreement between the two methods ranged from 85.7% to 100% for C. rectus,P. intermedia,
P. gingivalis,T. denticola,S. mutans and S. sobrinus. Lower agreement between the OralDisk
and the lab-based reference positives was observed for F. nucleatum (16/23 (69.6%) cases)
and Lactobacillus spp. (7/11 (63.6%) cases).
It is important to mention that each sample was analyzed with three technical repli-
cates with the lab-based PCR reference compared to one with the OralDisk to simulate
the POC workflow. In three cases within group (d), the lab-based PCR reference did not
give identical results for the triplicates. In two cases, two of three repeats for F. nucleatum
were detected positive and in one case, one of three repeats for Lactobacillus spp. was
detected positive. This may imply that these particular PCR assays in those samples were
close to the limit of detection for the lab-based reference, which would explain why they
were missed by the OralDisk. Finally, the OralDisk detected A. actinomycetemcomitans
in only one of the four lab-based reference positive samples. In line with data from the
preceding clinical study, this species was not often detected among the samples, and when
detected, it was often associated with very low levels of target genome equivalents [
22
].
This species also did not appear to play any discriminatory role between the healthy, caries
and periodontitis groups in the aforementioned study and for the recruited age groups [
22
].
However, A. actinomycetemcomitans was included in the panel because it may play a role in
cases where early onset periodontitis (i.e., younger patient ages than usual) was suspected.
A possible source of disagreement between the two methods may be the different
approaches for bacterial lysis, DNA extraction and purification prior to PCR amplifica-
tion. The lab-based reference method used an enzymatic lysis methodology (lysozyme,
mutanolysin, proteinase K enzymes [
22
]) with prolonged incubation times, followed by
column-based purification. For the OralDisk, mechanical lysis was performed ex situ
using a hand-held bead-beating device (Terralyzer, Zymo Research, USA), followed by a
magnetic bead-based bind-wash-elute protocol [
31
] for DNA extraction and purification on
the disk. Enzymatic or mechanical lysis may be more efficient, depending on the cell wall
properties of certain bacterial species—the OralDisk panel included both Gram-positive
and Gram-negative bacteria (Section 2.2).
Discrepancies in DNA extraction efficiency may also be expected between the different
approaches (column versus bead-based), as well as between different test devices. These
factors may have an impact on the detection of low abundances of bacteria (i.e., higher
Cq values). Consequently, targets that are close to the limit of detection of the OralDisk
(in its current configuration) may still be detectable by the lab-based reference method.
This could partly account for discrepancies between the two datasets where the lab-based
reference, but not the OralDisk, appeared to detect certain bacterial species more frequently.
Biosensors 2021,11, 423 10 of 21
3.3. Performance Comparison between the OralDisk and the Commercial iai PadoTest
The iai PadoTest (iai PadoTest, Institut für Angewandte Immunologie IAI AG, Zuchwil,
Switzerland), a commercially available system for the detection of periodontal pathogens,
was used to analyze 18 out of the 24 samples. We compared the data obtained by the iai
PadoTest and the OralDisk for these 18 samples and for five species per sample (as not all
ten species were shared between these two methods). To enable a direct interpretation of the
results, we only compared the qualitative outcomes of the methods (i.e., presence/absence
of the target bacteria), as the OralDisk in this study did not provide quantitative values for
bacteria concentrations. The results from the OralDisk and iai PadoTest are summarized in
Table 2. The iai PadoTest positively detected 28 of the microbial target assays, while the
OralDisk detected the same 28 and 33 more, thus giving 61 in total. Possible explanations
for this discrepancy in detection between the two methods may be the different molecular
identification principles and/or assay protocols used [
22
,
27
]. In fact, the iai PadoTest is
designed to examine gingival crevicular fluid (GCF). However, for better comparability
with the lab-based reference and the OralDisk we used a modified protocol based on saliva,
as described and discussed previously [22].
3.4. Comparison of Cq Performance
The performance comparison between the OralDisk and the lab-based reference
(
Table 1
) does not consider the Cq values of the two methods. In this section, the Cq values
of group (a) (Section 3.2) are therefore shown as scatter plots, in order to observe whether
one of the two methods exhibited any trend in Cq for all bacteria in all samples, as well
as for all bacteria in each clinical diagnosis group (Figure 5). Each data point corresponds
to the detection of a specific bacterial species in a specific sample with both methods.
The y-axis error bars are derived from the standard deviations of triplicate (or in some
cases duplicate) measurements made with the lab-based reference PCR method [
22
]. The
calculation of such standard deviations was not possible for the x-axis (OralDisk PCR), as
each sample was tested with only one OralDisk cartridge.
The diagonal line y = x is shown in the scatter plots simply to assist the observation
and assessment of whether the OralDisk or the lab-based reference method show any
trend in specific Cq areas. Data points above the y = x line mean that for a specific
measurement, Cq(Lab-reference) > Cq(OralDisk), while data points below the y = x line
mean that Cq(OralDisk) > Cq(Lab-reference). Interestingly, it appears that for the healthy
group, more data points lie below the y = x line, while for the caries and periodontitis
groups and for all samples combined, the number of points above and below the
x=y
line appear to be balanced (Table 3). When deriving a linear fit trendline, the slope of
Cq(Lab-reference)/Cq(OralDisk) is very similar between the total, healthy, caries and
periodontitis groups (0.34, 0.38, 0.32, 0.36, respectively). This is an indication that the
relation between the two methods is consistent and independent of the nature of the three
groups included in the study. From qualitative observation, there seems to be a center of the
scatterplot clusters at Cq ~ 24. Across the area where the Cq is higher than ~24 (i.e., at lower
bacterial concentrations), the OralDisk method tends to generate positive signals at higher
Cq values than the lab-based reference. This further supports the hypothesis expressed
in
Section 3.2
, namely that lower bacterial concentrations that can still be detected by the
lab-based reference may be missed by the OralDisk. Conversely, across the area where Cq
values were lower than ~24 (i.e., at higher bacterial concentrations), increasing PCR signals
tend to be observed earlier for the OralDisk.
Biosensors 2021,11, 423 11 of 21
Table 1.
Comparison of the lab-based reference versus the OralDisk for positive/negative results. Each number in the table denotes the number of times an assay target was detected in the
OralDisk and lab-based reference experiments. ‘Ch.1–Ch10’ refers to the numbering of the respective OralDisk chamber where each assay target was detected. ‘Pos.’ and ‘Neg.’ are
abbreviations for ‘positive’ and ‘negative’, respectively.
Ch.1 Ch.2 Ch.3 Ch.4 Ch.5 Ch.6 Ch.7 Ch.8 Ch.9 Ch.10
A. actinomycetemcomitans C. rectus F. nucleatum P. intermedia P. gingivalis T. forsythia T. denticola Lactobacillus spp. S. mutans S. sobrinus
Number of Pos.,
Neg. over Total
Assay Targets
Lab-reference Pos. 4 23 a23 a24 22 a22 a15 11 24 7 175/234
Lab-reference Neg. 20 0 0 0 0 0 9 13 0 17 59/234
(a) OralDisk Pos.
and Lab-reference Pos. 1 21 16 22 22 22 15 7 22 6 154/175
(88.0%)
(b) OralDisk Pos.
and Lab-reference Neg. 0 0 0 0 0 0 2 5 0 0 7/59
(11.9%)
(c) OralDisk Neg.
and Lab-reference Neg. 20 0 0 0 0 0 7 8 0 17 52/59
(88.1%)
(d) OralDisk Neg.
and Lab-reference Pos. 3 2 7 2 0 0 0 4 2 1 21/175
(12.0%)
a
In one disk, signals of C. rectus and F. nucleatum where not measured, while in two (other) disks, signals of P. gingivalis and T. forsythia were not measured. For reasons of comparison, these assays were excluded
from the lab-based reference.
Table 2.
Comparison between the iai PadoTest and the OralDisk. ‘Ch.1–Ch10’ refers to the numbering of the OralDisk chambers where each bacterium was detected. Grey shading denotes
the bacteria that are not included in the iai PadoTest panel. ‘Pos.’ and ‘Neg.’ are abbreviations for ‘positive’ and ‘negative’, respectively.
Ch.1 Ch.2 Ch.3 Ch.4 Ch.5 Ch.6 Ch.7 Ch.8 Ch.9 Ch.10
A. actinomycetemcomitans C. rectus F. nucleatum P. intermedia P. gingivalis T. forsythia T. denticola Lactobacillus spp. S. mutans S. sobrinus
Number of
Pos., Neg.
Assay Targets
OralDisk Pos. 1 16 16 16 12 61
iai PadoTest Pos. 0 7 8 3 10 28
OralDisk Neg. 17 2 0 0 6 25
iai PadoTest Neg.
18 11 8 13 8 58
Biosensors 2021,11, 423 12 of 21
(Benchtop
vs
OralDisk)
b
2
2
3
3
(Benchtop
vs
OralDisk)
b
7
(p )
q
(
)
b
7
7
7
(p )
b
Figure 5.
Scatter plots correlating the Cq values from the OralDisk and the lab-based reference for all bacteria: (
a
) in all
three groups; (
b
) in the healthy group; (
c
) in the caries group; and (
d
) in the periodontitis group. Similar scatter plots for
each individual bacterium are depicted in the Supplementary Figure S2.
As each OralDisk analyzes the presence/absence of ten bacteria simultaneously, the
scatter plots in Figure 5represent the entire panel and contain the collective information
from all bacteria. In order to investigate whether some individual bacteria exhibit any
specific trends, we composed the scatter plots for each bacterium for all clinical diagnosis
groups. From Supplementary Figure S2, we observe different tendencies for some bacteria
(quantitatively summarized in Table 3). For example: (i) C. rectus,P. gingivalis and T. forsythia
tend to appear in the Cq(Lab-reference) > Cq(OralDisk) area (i.e., the OralDisk amplification
Biosensors 2021,11, 423 13 of 21
curves yielded earlier Cq values than the lab-based reference); (ii) S. mutans tends to appear
in the Cq(Lab-reference) < Cq(OralDisk) area (i.e., the OralDisk amplification curves yielded
later Cq values than the lab-based reference); and (iii) there is no trend for F. nucleatum,
P. intermedia and T. denticola.
Table 3.
Number of cases where the Cq(Lab-reference) was higher or lower than the Cq(OralDisk). Calculations are based
on the scatter plots in Figure 5. The bacteria A. actinomycetemcomitans,Lactobacillus spp. and S. sobrinus are given only
indicatively as their total cases were very few.
Number of Cases with
Cq(Lab-Reference) > Cq(OralDisk)
Number of Cases with
Cq(Lab-Reference) < Cq(OralDisk)
TOTAL
Cases
For all bacteria in all groups 80 51.9% 74 48.1% 154
For all bacteria in healthy group 12 35.3% 22 64.7% 34
For all bacteria in caries group 41 55.9% 30 44.1% 68
For all bacteria in periodontitis group
27 57.7% 22 42.3% 52
For A. actinomycetemcomitans in
all groups 0 0.0% 1 100.0% 1
For C. rectus in all groups 15 71.4% 6 28.6% 21
For F. nucleatum in all groups 8 53.3% 7 46.7% 15
For P. intermedia in all groups 10 45.5% 12 54.5% 22
For P. gingivalis in all groups 17 77.3% 5 22.7% 22
For T. forsythia in all groups 16 72.7% 6 27.3% 22
For T. denitcola in all groups 8 50.0% 8 50.0% 16
For Lactobacillus spp. in all groups 1 14.3% 6 85.7% 7
For S. mutans in all groups 4 18.2% 18 81.8% 22
For S. sobrinus in all groups 1 16.7% 5 83.3% 6
We further analyzed the Cq values from the OralDisk and the lab-based reference
and mapped their distributions per bacterial species for all three clinically diagnosed
groups (Supplementary Figure S1). It should be noted that due to the small number of
measured samples, clinical conclusions cannot be extracted from the results per se. From the
descriptive graphical representation, it can be observed that for P. intermedia,
P. gingivalis
and T. forsythia (and possibly also T. denticola, although N = 2 for the OralDisk), the
boxplots would tend to be distinguishable (especially if/when a higher number of samples
were tested), even though there are overlapping standard deviations. In cases where
testing a higher number of samples would lead to a smaller standard deviation, these four
bacteria would possibly be the first ones that the OralDisk would detect as differentiators
between clinical diagnosis groups. Within this small-scale study, the tendency towards
differentiation seems to be stronger with the OralDisk than with the lab-based reference,
as the median lines of the OralDisk results for the different diagnosis groups seem to be
further apart (even though not necessarily lower) than those of the lab-based reference,
especially between the healthy and the caries groups (actual median values given in
Supplementary Table S2
). Indicative of this (and being aware of the small number of
samples for thorough statistical analysis), for T. forsythia, the p-value between the healthy
and the caries groups was 0.033 with the OralDisk and 0.29 with the lab-based reference,
and between the healthy and the periodontitis groups, it was 0.057 with the OralDisk and
0.29 with the lab-based reference (Section 2.8).
3.5. Overall Evaluation of the OralDisk
The development and implementation of chair-side molecular diagnostics in the field
of oral health lags behind many other fields of healthcare, including infectious diseases such
Biosensors 2021,11, 423 14 of 21
as respiratory tract, bloodstream and gastrointestinal infections, for which point-of-care or
near-patient systems are commercially available or at the product development stage [
45
].
However, the socioeconomic burden of oral diseases is high, with expenditure for the
treatment of dental diseases reaching EUR 90 billion and additional productivity losses
of over EUR 50 billion in EU member states in 2015 [
46
]. Furthermore, the documented
relationship between periodontal and systemic diseases [
8
,
47
50
] has started to raise
awareness of the importance of oral health and especially of early diagnosis, prevention
and post-treatment monitoring.
Table 4summarizes some existing technologies, together with the bacterial pan-
els they detect. A commercial test for the biomarker-based detection of periodontitis is
available from Dentognostics GmbH (PerioSafe
®
) [
51
]. It detects a single protein marker,
namely the active matrix metalloproteinase-8 (aMMP-8) [
52
54
] but no bacteria per se.
Furthermore, although the molecular-based detection of oral bacteria has been reported by
MyPerioPath
®
[
55
], HR5
TM
(High Risk Pathogen Test from Direct Diagnostics [
56
]) and
iai PadoTest [
27
], all these methods are not chair-side-compatible but laboratory-based.
Thus, the samples need to be transported to a laboratory and the results are only avail-
able after some days, whereas the OralDisk delivers results in <3 h. The PerioSafe
®
and
PerioPOC
®
[
57
,
58
] require only 5 and 20 min, respectively, due to their lateral flow config-
uration. However, the panels of both tests are limited: for the former, to a single protein
biomarker; for the latter, to five periodontitis pathogens. Additionally, none of the methods
in Table 4detects caries-related bacteria but focus only on periodontitis-associated bacteria.
Interestingly, in terms of throughput, all chair-side technologies test one sample per run.
For the lab-based technologies, the number of samples tested per run may depend on the
logistics of the particular laboratory. The overarching features of the OralDisk platform
compared to the listed systems are that it detects ten major caries- and periodontitis-related
bacteria simultaneously from non-invasively collected whole saliva using molecular-based
detection and a chair-side compatible system.
In terms of specimens, some of the methods listed in Table 4use paper points from
periodontal probing (iai PadoTest, PerioPOC®). However, paper points have the inherent
disadvantage that they are invasive and only reflect the local bacterial distributions of
specific tooth pockets [
12
,
13
]. Saliva is an increasingly popular candidate for analysis as
it is easy to collect in tubes even in milliliter volumes and its collection is non-invasive
in nature [
59
,
60
]. The OralDisk requires 400
µ
L of saliva (compared to 3–4 drops of oral
rinse for PerioSafe
®
and 160
µ
L of lysis solution for the immersion of paper points of
PerioPOC
®
, with 20
µ
L of this volume being added to the lateral flow chip). For the
lab-based methods of iai PadoTest, MyPerioPath
®
and HR5
TM
the required volume is
not known. In any case, the availability of large amounts of saliva makes the required
volume of this specimen type less critical in terms of the test to be selected. Non-oral-
related diagnostics have also attempted to use saliva to detect biomarkers or nucleic
acids in diseases such as COVID-19, type 2 diabetes mellitus, cardiovascular diseases and
Alzheimer’s disease
[6167]
. The specimens used in the current study were whole saliva
samples derived from recruited individuals of diverse oral health background (healthy,
caries, periodontitis) during a clinical study [
22
]. Compared to spiked samples with known
compositions, this methodology offers decisive advantages: (i) the oral flora in healthy and
diseased individuals is best represented in saliva, since it already contains many of the
bacteria to be examined and (ii) for POC use, it is essential to ensure that the OralDisk is
compatible with the physical, biological and chemical properties of the natural matrix, i.e.,
whole saliva and not any artificial saliva matrix. Indeed, the compatibility of the LabDisk
with this complex matrix paves the way for applicability of the platform in areas that can
make use of this abundant and easy-to-acquire specimen.
Biosensors 2021,11, 423 15 of 21
Table 4.
List of basic performance characteristics for a range of molecular-based methods of oral health assessment. ‘LFT’ stands for ‘Lateral Flow Test’. Bacteria that are not included in the
panels of some technologies are marked with ‘X’.
Characteristics PerioSafe®iai PadoTest MyPerioPath®PerioPOC®HR5TM OralDisk
Saliva sampling Yes
(Oral rinse)
No
(Probe) Yes No
(Probe) Yes Yes
Chair-side compatible Yes No
(Requires lab)
No
(Requires lab) Yes No
(Requires lab) Yes a
Quantitative capacity Yes/No
(LFT) b
Yes
(qPCR)
Yes
(qPCR)
No
(LFT) c
Yes
(qPCR)
Yes
(qPCR)
Caries panel included No No No No No Yes
Number of bacteria in panel, tested per run None
(detects aMMP-8 biomarker) 6 11 5 5 10
Bacteria panel
Aggregatibacter actinomycetemcomitans - X X X X X
Tannerella forsythia - X X X X X
Porphyromonas gingivalis - X X X X X
Treponema denticola - X X X - X
Prevotella intermedia - X X X X X
Filifactor alocis - X - - - -
Fusobacterium nucleatum - - X - X X
Campylobacter rectus - - X - - X
Capnocytophaga species (gingivalis, ochracea, sputigena) - - X - - -
Streptococcus mutans - - - - - X
Streptococcus sobrinus - - - - - X
Lactobacillus spp. - - - - - X
Peptostreptococcus micros - - X - - -
Eikenella corrodens - - X - - -
Eubacterium nodatum - - X - - -
a
Once the mechanical lysis of bacteria is transferred from the Terralyzer into the OralDisk [
68
].
b
Lateral Flow Test (protein detection) [
50
53
]. Visual readout provides qualitative analysis. Quantitative analysis
requires a processing instrument. cLateral Flow Test (nucleic acid hybridization). Visual readout provides qualitative analysis [58].
Biosensors 2021,11, 423 16 of 21
An outstanding advantage of the OralDisk is the automated sample processing and
detection of the assay targets. The system is based on a microfluidic platform that integrates
all the necessary biochemical reagents and operations in a protocol requiring minimal and
simple hands-on work. The only short manual step is the homogenization of whole saliva
using a bead-beating hand-held device (Terralyzer, Zymo Research, USA), which combines
homogenization and bacterial lysis in one step (saliva bead-beating has also been previ-
ously reported as a lysis method for lab-based downstream analysis [
69
,
70
]). Even in such
a configuration, the hands-on work was estimated to be only 10 min, including the ex situ
saliva homogenization and mixing with the control bacterium S. marinus, the pipetting
into the disk, and the insertion of the latter into the processing instrument. This time is
far shorter than the manual sample preparation time (at least 1.5 h) that is required prior
to a laboratory-based PCR. Additionally, previously published work by the authors has
already demonstrated the use of an on-disk microfluidic unit operation for saliva homog-
enization [
68
] (as a pre-analytic approach for downstream protein analysis), which uses
disk-integrated magnets actuated by magnets above the disk [
33
]. This disk-compatible
approach is planned to be tested in the future as an in situ lysis and homogenization step. It
will be integrated with the DNA purification module and will thus replace the only manual
step (i.e., bead-beating with the mobile Terralyzer device) of the current OralDisk protocol.
Further automation can be achieved if the aforementioned in situ mechanical lysis step is
combined with downstream dilution and direct amplification (after biochemical optimiza-
tion), without the need for a bead-based bind-wash-elute protocol. This modification is
expected to reduce the total time-to-result by approximately 0.5 h. In addition, a drastic
reduction in the time-to-result is also planned to be achieved by means of a new LabDisk
Player that will implement contact heating using Peltier elements instead of air heating.
This method is expected to reduce the PCR time from ~2 h to < 0.5 h, thereby resulting in
an estimated total time-to-result of <1 h.
The major feature of automation of the OralDisk workflow leads also to a drastic
reduction of hands-on work and time that is required by laboratory personnel, which can
result in a reduction of the overall ‘hidden costs’ of laboratory workflows. Even though we
cannot assess the end-user transfer price of the OralDisk (as it is currently at development
stage), the actual cost-driver aspects have been identified, as well as the actions that need
to be taken at the product development stage, e.g., re-assignment from thermoforming
to injection molding fabrication method (for manufacturing of hundreds of thousands
of OralDisk cartridges); screening for more cost-effective polymeric cartridge materials
and the complete automation of the entire manufacturing workflow. In any case, the
price of such a diagnostic system should not be compared with single-biomarker or single-
parameter detection systems, as the OralDisk offers an increased range of information for
a high number of pathogens and for both periodontitis and caries diseases. In fact, the
realistic goal for the OralDisk is that the cost per pathogen tested becomes significantly
lower than that using a laboratory test.
The analytics were performed on a batch of samples sufficiently representative to allow
the platform to demonstrate its performance and applicability for the detection of a broad
panel of ten Gram-negative and Gram-positive bacterial species. As previously mentioned,
this means that both periodontitis and caries can be monitored simultaneously on a single
OralDisk. Periodontitis has been mainly associated with Gram-negative anaerobic bacteria,
while caries has been mainly associated with Gram-positive carbohydrate-fermenting bac-
teria [
71
]. The OralDisk contains one of the broadest panels available for bacterial detection
among comparable systems (Table 4). This is achievable due to the multiple reaction
chambers (#7, Figure 2) that enable geometric multiplexing. The level of multiplexing
can be further increased by multiple wavelength detection in the same chamber (color
multiplexing). For example, in this publication the Gram-positive bacterium S. marinus
was included as a process control and underwent the same lysis, extraction, purification
and amplification processes as the oral bacteria present in the test sample. S. marinus
primers/probes were included together with the bacteria-specific primers/probes in each
Biosensors 2021,11, 423 17 of 21
reaction chamber, providing an indication that the internal biochemical and microfluidic
processes had been correctly implemented. In the disks that we tested, there was no
case where the S. marinus was not detected, thereby confirming qualitatively that the
sample-to-answer process functioned successfully. The simultaneous detection of several
bacteria, including the process control, within a single sample is a major achievement of
the platform, which until now had demonstrated its capability for infectious diseases that
are associated with either one or two pathogens, such as sepsis [
18
], tropical infections [
20
]
and respiratory tract infections [19,21].
The ~90% agreement between the OralDisk and the lab-based reference amongst
targets detected as positive and negative indicates that the OralDisk platform may be
suitable for accurate molecular-based oral bacteria detection. Any differences could be
attributable to the different lysis, extraction and/or test device approaches utilized by
the two methods. An increase in the test sensitivity of the OralDisk might be achievable
using a pre-amplification step, which in a past application of the LabDisk technology
was shown to detect down to a few bacteria/mL [
18
]. However, high sensitivity may
not be particularly crucial for oral health screening purposes. For example, the oral
microbiota is diverse in both healthy and diseased patients, including both commensals
and opportunistic pathogens. It is the dynamic changes over time of bacteria among the
oral microbiota, rather than their mere presence/absence, that drives the dysbiotic changes
that lead to oral disease [
72
]. The fact that the OralDisk has demonstrated its capacity to
analyze oral bacteria provides a basis upon which the platform can be implemented in
time-course studies associated with patient monitoring, similar to the rationale reported by
Paquéet al. [
73
]. In such future studies, we will have the opportunity to proceed to the
quantification of bacteria, using OralDisk-derived calibration curves to convert Cq values
into numeric bacterial loads.
The LabDisk and the corresponding customized processing device used in this work
are amenable to further performance improvements (such as inclusion of the lysis step on
the disk and the subsequent reduction of the time-to-result) and may be applicable as an
auxiliary tool for oral microbial screening in dental POC settings. Finally, for truly holistic
monitoring of oral health, bacteria-based microbiological examination in the OralDisk will
be combined with protein biomarker concentration monitoring. The LabDisk has already
been shown to be compatible with immunoassays by the running of a basic reaction (with-
out detection) as a proof-of-principle demonstration [
74
]. A newly developed and tested
bead-based immunoassay with oral biomarkers [
75
] will enable an immunoassay disk to be
run in the same instrument as the PCR disk. This will increase the interoperability between
immunological and microbiological diagnostic outputs using this platform [
76
]. Subse-
quently, by using combined computational technologies and the OralDisk device, new di-
agnostic predictive models of disease development and progression may be generated [
73
].
Such developments will enable evidence-based pre- and post-treatment monitoring of a
range of oral (OralDisk) and non-oral (application-specific LabDisk) diseases.
4. Conclusions
This technical feasibility study demonstrated the ability of the OralDisk to detect a
broad range of seven periodontitis- and three caries-related bacteria from whole saliva
samples in < 3 h, using an automated platform for molecular-based detection. Importantly,
the platform was proven to be compatible with saliva as a sample matrix for the first
time, which paves the way for (i) ‘modernization’ of contemporary oral health by using a
molecular-based diagnostic tool for saliva (instead of probe-based) close to the chair-side
practice, acting supportively to ‘traditional’ methods and (ii) implementation in further
areas of non-invasive saliva-based diagnostics. Compared to the lab-based reference test,
the OralDisk results showed ~90% agreement amongst targets detected as positive and
negative. We observed that higher levels of bacteria (Cq values < 24) generally resulted
in lower Cq values with the OralDisk compared to the lab-based reference method. This
was primarily observed for C. rectus, P. gingivalis and T. forsythia. On the other hand, lower
Biosensors 2021,11, 423 18 of 21
bacterial levels (Cq values > 24) were mostly detected later with the OralDisk compared to
the lab-based reference method. This was primarily observed for S. mutans.
Optimization of the platform towards automation and reduction of the time-to-result
will further increase its potential for adoption at the chair-side. The future goals are to
apply the OralDisk platform in larger clinical studies and to determine its clinical potential
in (i) providing early detection/prevention of oral diseases before they are detectable with
a conventional clinical examination or radiographic methods and (ii) monitoring progress
during and after periodontal treatment by providing quantitative information on changes
in bacterial load. Furthermore, such a diagnostic system could potentially contribute to
more informed decision making regarding the prescription of oral antibiotics, while also
acting as an early warning system for underlying systemic diseases [
77
] such as diabetes
and cardiovascular diseases, which have previously been correlated with periodontitis.
Supplementary Materials:
The following are available online at https://www.mdpi.com/article/
10.3390/bios11110423/s1, Table S1: Raw experimental data (Cq values) from the OralDisk and the
lab-based reference method, as well as qualitative data from the iai PadoTest. Figure S1: Boxplots
of the Cq values for each bacterium detected by the OralDisk and the lab-based reference. Table S2:
Median and interquartile range (IQR, describing the middle 50% of values when ordered from lowest
to highest) of the Cq values from the OralDisk and lab-based reference measurements. Figure S2:
Scatter plots correlating the Cq values from the lab-based reference and the OralDisk for all bacteria
in the healthy, caries and periodontitis groups.
Author Contributions:
Conceptualization, D.B., B.J., R.Z., C.H., J.R.P., P.N.P., F.J.W., N.B., G.N.B. and
K.M.; methodology, D.B., B.J., M.S., J.L., R.Z., C.H., J.R.P., P.N.P., V.R., N.B., G.N.B. and K.M.; software,
S.H., C.H., J.S.J., A.S., A.M. and T.B.; validation, D.B., P.N.P., T.A., J.S.J., P.K., P.R.S., T.T., F.J.W. and
V.R.; formal analysis, D.B., C.H., P.N.P., J.S.J., A.S., V.R., K.B., G.N.B. and K.M.; investigation, D.B.,
B.J., M.S., J.L., M.R., S.H., C.H., P.N.P., J.S.J., W.E.K. and K.B.; resources, M.R., R.Z., P.N.P., T.A., J.S.J.,
P.K., P.R.S., T.T., F.J.W., V.R., D.S. and M.K.; data curation, C.H., J.S.J. and A.S.; writing—original
draft preparation, D.B., G.N.B. and K.M.; writing—review and editing, D.B., B.J., M.S., J.L., S.H., N.P.,
F.v.S., R.Z., C.H., J.R.P., P.N.P., T.A., J.S.J., P.K., P.R.S., T.T., F.J.W., W.E.K., J.P.H., V.R., A.M., T.B., M.K.,
K.B., N.B., G.N.B. and K.M.; visualization, D.B., S.H., C.H., J.R.P., W.E.K. and K.M.; supervision, N.P.,
F.v.S., R.Z., J.R.P., P.N.P., T.A., P.R.S., T.T., J.P.H., D.S., N.B., G.N.B. and K.M.; project administration,
R.Z., J.R.P., P.N.P., T.A., T.T., D.S., M.K., N.B., G.N.B. and K.M.; funding acquisition, J.R.P., P.N.P., T.A.,
F.J.W., J.P.H., A.M., M.K., N.B., G.N.B. and K.M. All authors have read and agreed to the published
version of the manuscript.
Funding:
This project has received funding from the European Union’s Horizon 2020 research and
innovation programme under grant agreement No 633780 (‘DIAGORAS’ project), as well as the
KI/SLL Styrgruppen för Odontologisk Forskning (SOF) Dnr. 4-823/2019. The article processing
charge was funded by the Baden-Wuerttemberg Ministry of Science, Research and Art and the
University of Freiburg in the funding programme Open Access Publishing.
Institutional Review Board Statement:
The study was conducted according to the guidelines of the
Declaration of Helsinki and approved by the local Swiss Ethics Committee with BASEC-No. 2016-
00435 and the date of approval: 9 January 2016.
Informed Consent Statement:
Informed consent was obtained from all the subjects involved in
the study.
Data Availability Statement: Data is contained within the article or in the supplementary material.
Acknowledgments:
The authors would like to acknowledge the Hahn-Schickard Lab-on-a-Chip
Foundry Service for LabDisk production. We would like to thank Helga Lüthi-Schaller for her
excellent laboratory and technical support during the experiments of the clinical study. We would
also like to thank Jennifer Berkman and Claudia Steffen from Thermo Fisher Scientific, who greatly
assisted this research by providing access and tailoring a custom TaqMan
®
Lyophilized 1-Step qPCR
Master Mix towards the needs of this project. We are immensely grateful for their constructive and
prompt support.
Biosensors 2021,11, 423 19 of 21
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
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... Lyophilization or freeze-drying is often used to store proteins (alone, i.e., without beads) due to the good stabilization achieved with this method, even though it is costly and technologically complex [61]. Concerning beads alone, we have demonstrated protocols for pre-storage in the case of magnetic beads for nucleic acid extraction and purification [62][63][64]. However, the BFPD-IA that is integrated within the ImmunoDisk involves protein-coupled magnetic and fluorescent beads. ...
... These conditions simplify the fluidic integration and reduce the footprint on disk. Importantly, the ImmunoDisk was processed on a device, the LabDisk Player 1 functional model, which was developed for the automation of nucleic acid amplification technologies (NAATs, e.g., PCR and isothermal methods) and for which different applications have already been shown (e.g., respiratory tract infections [64], tropical infections [63], oral diseases [62] and vector analysis in mosquitos [76]), without any changes to the device hardware. The compatibility of the ImmunoDisk with this NAAT device is primarily due to the protocol and the detection principle of the BFPD-IA, as well as the adaptation of the microfluidic structures. ...
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