Biosensors & Bioelectronics 13 (1998) 757–762

DNA biosensors based on Peptide Nucleic Acid (PNA) recognition

layers. A review1
*
Joseph Wang
Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, NM 88003, USA

Received 20 January 1998; accepted 23 April 1998

Abstract

Peptide nucleic acid (PNA), originally developed as a gene-targeting drug, has demonstrated remarkable hybridization properties
towards complementary oligonucleotides. Biosensors based on replacement of the DNA recognition layer with a PNA one, offer
greatly improved distinction between closely related sequences, as well as several other attractive advantages. The present review
discusses the unique structural, hybridization, and recognition features of PNA probes, along with the opportunities accrued from
the use of PNA recognition layers in DNA diagnostics.  1998 Elsevier Science S.A. All rights reserved.

Keywords: DNA biosensors; Peptide nucleic acid; Hybridization; Genosensors; Mismatch discrimination

1. Introduction (Mikkelsen, 1996) have been used to convert the

The use of nucleic acids recognition layers in
biosensor design represents a new and exciting area in
analytical chemistry (Wang et al., 1997a). Such recog-
nition layers add new and unique dimensions to the
arsenal of modern biosensors. In particular, DNA
hybridization biosensors offer considerable promise for
obtaining sequence-specific information in a faster, sim-
pler and cheaper manner compared to traditional
hybridization assays. Hence, such devices possess great
potential for numerous applications, ranging from
decentralized clinical testing, to environmental monitor-
ing, food safety and forensic investigations.
The basis for these nucleic-acid hybridization devices
is the DNA base pairing, namely the strong interaction
between two complementary nucleic acid strands.
Accordingly, such devices rely on the immobilization of
a single-stranded (ss) DNA molecule (the “probe”) for
hybridizing with the complementary (“target”) strand in
a given sample (containing also non-complementary
nucleic acids). Various transduction modes, including
optical (Piunno et al., 1995) and electrochemical
* Fax: 001 505 646 6033
1
This paper was a finalist for the Biosensors & Bioelectronics
Award for the most original contribution to the Congress.
hybridization event into a measurable electrical signal in
connection with a proper indicator (that interacts differ-
ently with the surface-confined single- and double-
stranded DNAs).
As with other types of biosensors, high selectivity is
crucial for the success of DNA biosensors. The speci-
ficity of DNA hybridization devices (i.e., their ability to
respond to the target sequence in the presence of non-
complementary strands) depends primarily on the selec-
tion of the probe, and secondary upon the hybridization
conditions (mainly the temperature). To date, however,
most DNA biosensors are not capable of selectively dis-
criminating against single-base mismatches, as desired,
for example, for the detection of disease-related point
mutations. The ability to recognize a change in a single
nucleotide thus represents a major challenge for geno-
sensor technology. Accordingly, a drastic approach is
desired to impart higher selectivity onto nucleic-acid
biosensors. Remarkable sequence specificity has been
achieved recently using PNA recognition layers (Wang
et al., 1996). This article reviews the structure and
unique hybridization properties of these synthetic DNA
analogs, along with recent developments of PNA biosen-
sors and their prospects for future DNA diagnostics.

0956-5663/98/$—see front matter  1998 Elsevier Science S.A. All rights reserved.
PII: S 0 9 5 6 - 5 6 6 3 ( 9 8 ) 0 0 0 3 9 - 6
758 J. Wang / Biosensors & Bioelectronics 13 (1998) 757–762

2. PNA structure to traditional oligonucleotides. It was shown (Egholm

et al., 1993) that such hybridization to complementary
PNA was first described by Nielsen’s group in 1991 oligonucleotides obeys the Watson–Crick base-pairing
(Nielsen et al., 1991). It was originally designed (using rules with the PNA and DNA strands joined through
computer model building) as a reagent to specifically hydrogen bonds (Fig. 2); it was demonstrated also that
bind double-stranded (ds) DNA, using the triple-helix the thermal stability of the resulting PNA/DNA duplex
principle, for controlling gene expression in connection is essentially independent of the salt concentration in the
with the development of antisense and antigene thera- hybridization solution (Orum et al., 1995). Such inde-
peutic drugs. The amide-based synthetic chemistry and pendence of the ionic strength is the result of the
medicinal properties of PNA were reviewed recently uncharged nature of the PNA backbone.
(Hyrup and Nielsen, 1996). The neutral backbone also implies a lack of electro-
Knowledge of the PNA structure is essential for static repulsion between the PNA and DNA strands
understanding its remarkable hybridization behavior and (compared to that existing between two negatively-
DNA recognition action. PNA is an analog of DNA in charged DNA oligomers), and hence a higher thermal
which the entire negatively-charged sugar-phosphate stability of PNA/DNA duplexes. On average, the melting
backbone is replaced with a neutral “peptide-like” back- temperature (tm) of a PNA/DNA duplex is 1°C higher
bone consisting of repeated N-(2-aminoethyl)glycine per base pair compared to that of the corresponding
units linked by amide bonds (Fig. 1). The four natural DNA/DNA duplex (Hyrup and Nielsen, 1996). The ther-
nucleobases (i.e., adenine, cytosine, guanine, and
thymine) come off the backbone at equal spacing to the
DNA bases. Methylene carbonyl linkages connect the
bases to the central amine of the backbone. Thus, PNA
contains the same number of backbone bonds between
bases (i.e., six) and the same number of bonds between
the backbone and the bases (i.e., three), as in DNA. Such
structure is not prone to degradation by nucleases or pro-
teases, hence offering high biological stability (Demidov
et al., 1994). The unique structure and the resultant
hybridization properties of PNA (discussed below), open
up many important biological and diagnostic appli-
cations, not achievable with traditional oligonucleotides.

3. PNA hybridization properties

Owing to its neutral backbone and proper interbase

spacing, PNA binds to its complementary nucleic acid
sequence with higher affinity and specificity compared

Fig. 1. Chemical structure of PNA (top) and DNA (bottom), where Fig. 2. PNA–DNA recognition by Watson–Crick hydrogen bonding
B is the nucleobase. (Reproduced with permission from Nielsen and Haaima, 1997.)
 J. Wang / Biosensors & Bioelectronics 13 (1998) 757–762
mal stability of the PNA/DNA duplexes is strongly
affected by the presence of imperfect matches. Such
presence of mismatches in a PNA/DNA duplex is much
more destabilizing than a mismatch in a DNA/DNA
duplex. For example, a single base mismatch results in
15 and 11°C lowering of the tm of PNA/DNA and
DNA/DNA duplexes, respectively. This property of
PNA is responsible for the remarkable discrimination
between perfect matches and mismatches offered by
PNA probes, and makes them so attractive as oligonucle-
otide recognition elements in biosensor technology.
4. PNA biosensors
The unique properties displayed by solution-phase oli-
gomers can be extrapolated onto transducers surfaces in
connection with the design of DNA biosensors. The
advantages accrued from the use of PNA recognition
layers were realized first in our laboratory in connection
with electrochemical transduction of the PNA/DNA
hybridization process (Wang et al., 1996). For this pur-
pose, the PNA probe was immobilized by adsorption
onto the carbon-paste electrode transducer, and the for-
mation of the hybrid was detected by exposure to a sol-
ution of the Co(phen)33 + redox indicator. Fig. 3 shows
the response of the 15-mer PNA biosensor to increasing
levels of the DNA oligonucleotide target. Dotted lines
represent the response in the absence of the target. The
increased indicator peak area, upon its association with
the surface duplex, thus serves as the hybridization sig-
nal. Such chronopotentiometric transduction mode offers
a defined indicator signal along with a low back-
ground response.
This early study illustrated that the PNA-derived elec-
Fig. 3. Chronopotentiograms for the Co(phen)+3 3 indicator obtained at
the PNA-modified carbon paste electrode following a 5-min hybridiz-
ation in solutions containing increasing levels of the complementary
DNA target in 0.2 ppm steps. (Reproduced with permission from Wang
et al., 1996).
759
trochemical sensor offers a greatly improved distinction
between closely related sequences, and provides greater
latitude in the selection of experimental conditions,
including efficient hybridization in low ionic strength
solutions or at elevated temperatures, as well as the use
of short probes. The hybridization response of the PNA
biosensor was nearly independent of the ionic strength
and hybridization temperature over the 1–20 mM phos-
phate-buffer concentration and 22–50°C, respectively. A
detection limit of 10 pmol of the 15-mer oligonucelotide
target was observed following a 10-min hybridization.
Subsequent work (Wang et al., 1997b) illustrated the
utility of the PNA carbon-paste biosensor for the detec-
tion of specific mutation in the p53 gene, a mutation
related to various types of cancer. Challenging the PNA
biosensor with the single-base mismatch oligomer
resulted in a 3% error, as compared to a 91% error for
the DNA recognition.
Since the success of electrochemical (or other) PNA
sensing schemes depends in part on the immobilization
of the PNA probe onto the surface, it is essential to eluci-
date the interfacial behavior of PNA. As expected from
the electrical neutrality of the PNA backbone, the
adsorption of PNA onto charged carbon or mercury elec-
trodes differs greatly from that of DNA in terms of
potential dependence, surface packing and salt effect
(Fojta et al., 1997; Wang et al., 1997c). The strong
adsorption of PNA can be exploited for an effective pre-
concentration step, and combined with the electroactivity
of the nucleobases for developing highly sensitive elec-
trochemical stripping protocols for measuring trace lev-
els of PNA.
The use of quartz crystal microbalance (QCM) trans-
ducers can also benefit from the attractive properties of
PNA probes (Wang et al., 1997d). Such use of QCM
transducers offers an indicator-free direct detection of
DNA hybridization based on monitoring the decrease in
the resonance frequency of the crystal associated with
the mass increase at the surface during the hybridization.
A remarkable mismatch discrimination was observed at
the PNA-QCM biosensor in connection with the detec-
tion of a specific mutation in the p53 gene (Fig. 4). Such
improved specificity in the presence of a large excess of
the single-base mismatch (in comparison to PNA-modi-
fied carbon electrodes) is attributed to the formation of
a packed PNA layer, in connection with the self-
assembly of the thiol-derivatized PNA on the gold-
coated crystal. Such packing, coupled with the hydro-
philic character of the ethylene glycol linker, result also
in negligible non-specific adsorption effects. The selec-
tivity improvement was coupled to the use of short (15-
mer) probes and low ionic strength solutions. (It should
be noted, however, that the primary concern regarding
the length of the PNA probe should be the specificity
for a given application.) Further improvements in the
sensitivity of QCM/PNA devices may be achieved by
760 J. Wang / Biosensors & Bioelectronics 13 (1998) 757–762
Fig. 4. Frequency-time response of the QCM-PMA biosensor to mul-
tiple additions of 10 ppm of the 15-mer p53 DNA target (designated
as T) and 50 ppm of the 15-mer single base mismatch oligomer
(designated as M). (Reproduced with permission from Wang et al.,
1997d.)
increasing the hybridization capacity through the use of
multilayer PNA films or branched (dentritic) PNA pro-
bes.
Optical DNA biosensors are based on changes in the
optical properties of the interface associated with the
hybridization event. Surprisingly, the coupling of PNA
probes with fiber-optic transducers has received little
attention (despite the growing role of fiber-optics and
fiber-optic arrays for biosensing of DNA). The use of
PNA probes holds promise not only for enhancing the
specificity of these devices, but can be used for
developing new spectroscopic detection principles. In
particular, an elegant work from Oak-Ridge National
Laboratory demonstrated a non-labeling mass-spectro-
scopic hybridization detection based on monitoring the
phosphorous backbone of the DNA target (Arlinghaus
et al., 1997). Because the immobilized PNA probe does
not have any phosphorous, the phosphorous signal can
be used for detecting the hybridized DNA. The authors
reported an excellent discrimination between comp-
lementary and noncomplementary sequences, including
increased discrimination against single point mutations
in connection with arrays of PNA probes on a silicon
chip to produce the phosphorous image (Fig. 5).
Larger scale arrays of multiple PNA probes could
facilitate ultrafast DNA sequencing. Arrays of up to
1000 PNA oligomers of individual sequences were
recently synthesized on polymeric membranes (Weiler
et al., 1997). The array-bound PNA probes displayed
higher affinity to DNA targets than the corresponding
DNA oligonucleotides probes (although non-specific
DNA binding was also observed in connection to the
fluorescent detection).
Surface plasmon resonance (SPR) spectroscopy was
recently employed for studying the hybridization kin-
etics of PNA/DNA and PNA/RNA duplexes (Jensen et
al., 1997). The PNA was tethered to steptavidin at the
dextran/gold surface via a biotin linker. Such coupling
of PNA probes with the BIAcore technique should be
useful for in situ hybridization detection of unlabeled
DNA targets.
A new laser-based fluorescence technique utilizing
PNA probes, developed recently for single-molecule
detection of specific nucleic acid sequences in unampli-
fied genomic DNA (Castro and Williams, 1997), holds
great promise for biosensor applications. The method
involves the use of two PNA probes complementary to
different sections of the DNA target. The two probes,
each labeled with a different fluorescent dye, forms a
hybrid with the target DNA, that can be detected at two
different wavelengths.
5. Outlook
PNA has already established itself as an attractive rec-
ognition layer in DNA biosensor technology. The unique
structural and hybridization features of PNA make it
superior to DNA for use as a sequence-specific hybridiz-
ation probe, and open up exciting opportunities for DNA
diagnostics. Such unique properties of solution-phase
PNA can be extrapolated onto transducer surfaces in
connection with the design of DNA biosensors. While
offering greater mismatch discrimination, higher biologi-
cal stability, and operation over a wide range of
hybridization conditions (compared to their DNA
counterparts), PNA biosensors are still not ready for
large-scale decentralized testing applications. Such
applications would require improvements in the sensi-
tivity of PNA biosensors, and in their ability to recognize
mutations in large DNA fragments or in long PCR pro-
ducts, as well as proper attention to non-specific bind-
ing events.
Current development of various PNA derivatives,
based on the modification or extension of the PNA back-
bone or linker (Hyrup and Nielsen, 1996), holds great
promise for various sensing applications. Corresponding
structural-activity studies should contribute to the
judicious design of PNA derivatives with improved
hybridization potency and mismatch discrimination
(compared to the parent glycine PNA).
The drastically different structure and behavior of
PNA has important implications not only upon the
hybridization conditions, but also upon the detection and
regeneration steps. For example, suitable hybridization
indicators are also desired in view of the different bind-
ing affinities of small molecules with PNA–DNA and
DNA–DNA hybrids (Wittung et al., 1994). Built-in
(fluorescent or redox) labels, covalently attached to the
PNA oligomer, may offer an attractive alternative. New
detection schemes, relying on the different nature of the
 J. Wang / Biosensors & Bioelectronics 13 (1998) 757–762 761

17-mer PNA oligo which is

complementary to M13(-20)

17-mer PNA oligo which is

noncomplementary to M13(-20)

Relative
P Signal
0.040
0.035
6 0.030
0.025
5

0.020
4

7
m)

6
(m
3

5 0.015
on

4
2

0.010
siti

X-P 3
o

2
1

osi 0.005
Y-P

tion 1
(mm
0
0

Fig. 5. Sputter-initiated resonance ionization microprobe (SIRIMP) phosphorus image of a DNA oligonuclotide hybridized to a PNA probe of a
biosensor chip. (Reproduced with permission from Arlinghaus et al., 1997.)

PNA backbone, may lead to novel indicator-free stra-
tegies. For example, pulse or sinusoidal amperometry or
copper electrodes may be used for detecting the sugar
moiety of the hybridized DNA target (in the absence of
such moiety in PNA). Alternately, one can rely on the
electroactivity of the target guanine bases in connection
to the use of guanine-free (inosine-substituted) PNA pro-
bes. The high thermal stability of PNA–DNA duplexes
requires reassessment of the thermal and chemical regen-
eration conditions used for “removing” the bound target
DNA. Faster surface renewal may be achieved mechan-
ically in connection to polishable probe-confined com-
posite materials (Wang et al., 1998). In addition, it may
be possible to exploit the different charges of PNA and
DNA and the marked difference in their adsorption
behavior for minimizing non-specific DNA adsorption
via a judicious choice of the electrode potential (Fojta
et al., 1997) High-density PNA arrays should further
enhance the discriminative power of nucleic acid
hybridization tests, and would allow detection of mul-
tiple sequences in a single experiment. Coupling of these
microfabricated arrays with micromachined (on-chip)
PCR amplification units, and with compact easy-to-use
instruments, will greatly facilitate the realization of rou-
tine on-site DNA testing. These and other on-going
research and commercialization efforts will surely make
PNA-derived biosensors extremely useful tools in bioan-
alytical chemistry.
Acknowledgement
I gratefully acknowledge the financial support of the
NM Water Resources Research Institute. I would also
like to acknowledge the contributions of members of my
DNA-sensor research team—too numerous to list—to
the development of PNA-derived biosensors, and the
very fruitful collaboration with Professors P.E. Nielsen
and E. Palecek.
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