Research progress of near -infrared fluorescence immunoassay [621791]

Research progress of near -infrared fluorescence immunoassay

Abstract: Near-infrared fluorescence immunoassay ha s been widely studied in the bioanalytical
field. This review mainly introduces the basic principles of near -infrared spectroscopy and
near-infrared detection technology, and summarizes the properties, characteristics and recent
impro vement of optical properties and signal intensity of three near-infrared fluorescence probes
(i.e. organic fluorophores , quantum dots and rare earth com pound s). We describe the application s
of near -infrared fluorescence technology in immunoassay , and prospect the application potential
of lateral flow assay (LFA) based on this probe in the rapid detection of pathogen s. Our team
intends to establish a new platform which has highly sensitive near -infrared fluorescence
probe s(NIFP s) combined with portable and simple immunochromatographic test strips (ICTSs) for
rapid detection of food borne viruses. This will provide technical support for a rapid detection on
the port .

Practical applications
The applications of near-infrared fluorescence probes (NIFPs ) in immunological analysis and
clinically important biomarkers were also elaborated. Moreovere, NIFPs-based i mmunoassay
adaptable for rapid detection of foodborne pathogens was also forecasted. In 2017 , our team has
developed a n approach for detecting pathogen s such as Salmonella, Vibrio parahaemolyticus,
Vibrio cholerae and Listeria monocytogenes by near -infrared immunoassay .

Key words : near-infrared fluorescence probe s (NIFPs) immunochromatographic test strips
(ICTSs ) organic fluorophores quantum dots rare earth com pound s immunoassay

1 Introduction

The infrared spectroscopy is an electromagnetic spectrum between the visible r egion and the
microwave region . It is divided into three regions: near -infrared, mid -infrared, and far -infrared.
The near -infrared band is between 780-2526 nm spectral sections of the electromagnetic spectrum
(Siesler et al.,2008). Due to its high sensitivity and selectivity , fluorescence sp ectroscopy has a
wide range of applications in analytical chemi stry, especially in bioanalysis . Most biomolecules
have no fluorescence or weak fluorescence, and their detection sensitivity is low. In order to detect
them wit h high sensitivity, people use fluorescent labeling reagents or fluoregenic reagents to label
or derivatize the analytes. The formation of a covalent or non -covalently bound material with high
fluorescence intensity greatly decrease s the detection limit.
Fluorescent reagents such as fluorescein, rhodamine or phthalaldehyde (OPA) currently used for

Near-infrared fluorescence immunoassay

labeling or derivatization . They have high fluorescence quantum yields, but their maximum
absorpti on wavelength and fluorescence emission wavelength are mostly less than 600 nm. For
biological s amples, the sample matrix and some impurities also have light absorption or
fluorescence in this area. In addition, the effect of light scattering will often cause more serious
background interference, which limits the sensitivity of fluorescence analysis. Compared with the
conventional fluorescence ( em<600nm) detection, the light absorption or fluorescence intensity
of the biological sample matrix is small in the near -infrared fluorescence ( em>600nm) region .
Therefore, the background interference is grea tly reduced, and since the intensity of the scattered
light is inversely proportional to the fourth p ower of the wavelength . As the wavelength increases,
the Raman scattering rapidly decreases, and the scattering interference is also greatly reduced.
In re cent years, near -infrared fluorescent labeling reagents and detection techniques based on
diode lasers with compact structure, good stability and low price have higher sensitivity, and have
been used for near -infrared fluorescence immunoassay, flow cy tomet ry, fluorescence detection of
biological active substances in high performance capillary electrophoresis separation. At the same
time, with the continuous integration of various technologies, the development o f many rapid
detection devices such as laser fl uorescence, sensor s and immunodetection devices has promoted
the development of near -infrared fluorescent marker detection and analysis in the biological field.
NIR spectroscopy technology has many advantages which determine its wide application field.
NIR spectroscopy c ould be used for food quality analysis, such as researching the freshness of
agricultural products, testing fruit firmness and quality, detecting fruit sugar and acidity,
controlling the quality of baked products and detecting alcohol durin g alcohol fermentation in
food industry (Wang et al.,2011) . It also could detect c hanges in sugar content, prediction of
different meat characteristics, determination of nutrients in dairy products and identification of
the authenticity of edible oil . In agricultur e, it c ould be applied to crop quality analysis and
evaluation, crop variety resource ident ification and quality breeding and crop resistance index
analysis. Near -infrared spectroscopy has new developments in other fields such as microbiology.
Currently, most of the research objects are bacteria and yeast and a few objects are fungi and
algae.

2 Near -infrared fluorescent probe types

Near infrared fluorescence immunoassay is a novel lateral flow assay (LFA) that combines
near-infrared fluorescence probes (NIFPs) with immun oassay . In view of its outstanding
advantages of small background interference and strong tissue penetration, NIFPs have attracted
more and more attention in recent years. Due to the limited sensitivity of labele d probes based on
color signals such as colloidal gold , enzymes and electrochemical signal generation probes are
expensive and cumbersome to operate, and it is difficult to achieve one -step detection. NIFPs have
become one of the most popular signals m olecules are widely used in various bioanalytical fields
(Pyo & Yoo,2012) . NIFPs with emission spectra in the near -infrared region (wavelength 650~1 100
nm), which attracts attention in the field of analysis owing to their high signal -to-noise ratio and
ideal d etection sensitivity (Heise ,2002) . First, the biomatrix rarely fluoresces spontaneously in the
near-infrared region, allowing NIFPs -based assays to be protected from background fluorescence
interference. Second, because the intensity of the scattered ligh t is inversely proportional to the
fourth power of the wavelength, NIFPs with emitted light in the long -wave r egion are subject to

Near-infrared fluorescence immunoassay

its interference is small. The strong penetrability and small damage to biological tissues is another
major advantage of NIFP s, which has been widely us ed in non -destructive testing ( Alander et
al.,2013) and biography (Guo et al.,2014) .

2.1 Near -infrared fluorescent material
Although new forms of NIFPs are being synthesized, traditional organic fluorescent dyes are
still a main stream of current nea r-infrared fluorescent probes. Traditional organic fluorescent dyes
such as cyanine, rhodamine , and other thiazide organic dyes, are widely developed as NIFPs in
which cyanine dyes are most fav ored for good biocompatibility (Stoyanov , 2000) . Cyanine
dye-labeled biological samples mainly have non -covalent , covalent forms of
electrostatic/hydrophobic interactions and bioconjugates with some biomolecules through reactive
groups for labeling nucleic acids, proteins, immunoassays and contras t.
Lee et al. (Lee et al. ,2013) further improved and derivatized it using the DOFLA
(different -oriented fluorescence library approach), and obtained more than 40 NIFPs with
excellent physicochemical properties. The light stability of AZA396 was 60 times h igher than that
of BodipyFl. Researchers not only synthesize new near -infrared organic probes, in order to
better apply it to biological analysis, but also pay attention to the traditional near -infrared
fluorescent dye water solubility, quantum yield, che mical and light stability, biocompatibil ity
improvements in key traits (Fu et al.,2013) . Aggregation of dyes or dye combinations can lead to
severe quenching of fluorescence (Gruber et al.,2000) , and many studies have focused on
improving the water solubil ity of dyes. Since it was first discovered and reported by Waggoner et
al (Waggoner et al.,1993) in 1993, attachment of sulfonate groups to the aromatic ring is effective
in increa sing the water solubility of NI FPs. This conclusion was also confirmed by Ch eng et al.
(Cheng et al., 2015 ). The well -known commercialized near -infrared organic molecules Cy5.5 and
Cy7 from Amersham Biosciences are all sulfonate phthalocyanine dye structures. In addition,
studies have shown that the ideal water solubility can also be achieved by coating a hydrophobic
dye onto a single layer of hydrophilic phospholipi ds on the surface of liposomes (Chen et
al.,2007 ). In order to effectively increase the fluorescence intensity of dyes to determine trace
target analytes, researchers co ntinue to explore efficient signal amplification strategies. For
example, a large amount of fluorescent dye is wrapped in nanoparticles to form a near -infrared
nanoparticle probe with higher fluorescence intensity, which has been widely confirmed to
effect ively improve the detection signal intensity while improving the chemical and photosta bility
of the labeled molecule (Christian et al.,2007) . Furthermore , based on the surface plasmon
resonance of the metal nanostructure, the fluorescence intensity of the near-infrared fluorescent
dye can also be significantly improved. As studies have shown, by using rough metal surfaces
such as silver island films or gold nano -shells, the signal intensity of phthalocyanine green can be
increased b y 20 and 50 times respect ively (Malicka et al.,2007; Tam et al.,2007 ). In addition,
coating the nano -microspheres with a multi -polymer material coated with a near -infrared
fluorescent dye has been indicated to improve the biocompatibility of the dye. For example, Kim
et al. has improve d the compatibility of dyes with cells by encapsulating Cy5.5 in a hydrophilic
polymer. NIFP c ould be used to monitor the imaging changes of cell structure in early stage of
apoptosis (Kim et al.,2006 ) .

2.2 Near -infrared fluorescent quantum dot

Near-infrared fluorescence immunoassay

Quan tum dots (QDs) known as semiconductor nano -microcrystals, have been widely used in
bioanalysis and medical diagnostics recent ly as a new class of fluorescent probes due to thei r
excellent optical properties (Kairdolf et al.,2013). The fluorescence emission spectrum of this
probe with adjustable particle size and composition ensures its feasibility as a near -infrared
labeled probe. Near -infrared quantum dots refer to quantum dots with emission wavelengths
between 650 and 900 nm, and have the dual characteris tics of near -infrared and quantum dots.
Compared with traditional organic fluorescent dyes, QDs have unparalleled advantages such as
high quantum yield, stro ng anti -photobleaching ability and concentrated emission spectrum. As an
emerging biological probe, applications of quantum dots are still expanding in scope. However, it
cannot replace the traditional organic small molecule fluorescent probe, and can only be used as a
powerful supplement to the existing organic small molecule fluorescent probe. Biocomp atibility
remains to be further explored because of its potential toxicity to living tissue. Currently, quantum
dot-based chromatographic test strips have been widely used in food safety, environmental
monitoring, medical diagnosis and other fields (Berlin a et al.,2013; Zou et al.,2010; Gui et
al.,2014)

2.3 Near -infrared fluorescent rare earth complex
Complexes of rare earth elements (lanthanides) containing Nd 3+ , Er 3+ , Yb 3+ and Tm 3+ in the
near-infrared region have been widely developed in recent ye ars. ( Aita et al.,2010; Yu et al.,2007;
Korovin et al.,2010; Zhang et al.,2010) . Near -infrared fluorescent rare earth complexes have
unique advantages such as large stock' s displacement, long fluorescence lifetime, and no
photobleaching relative to NIFPs such as organic dyes and semiconductor nanocrystals (Comby et
al.,2007) . The use of free lanthanides is often hindered by the need for a photonic converter to
handle vibrational overtone spectroscopy induced by -OH, -NH and -CH due to th e low extinction
coefficient (Eliseeva et al.,2010) . In order to overcome the difficulties , many researchers are
committed to the further optimization of NIF lanthanides. For ex ample, Foucault -Collet et al.
(Foucault -Collet et al.,2013) developed a unique NIF rare earth meta l-organic frameworks (MOFs)
that encapsulate a large number of NIF -emitting Yb 3+ ions with the sensitizer phenylenevi nylene
dicarboxylate ( PVDC ) in a small volume. This structure not only provides a new way for
sensitization and protection of lanthanides , but also greatly improves the detection sensitivity due
to the increase in the number of probes carried per unit volume. In addition, incorporation of rare
earth e lements into laser materials (Duan et al.,2006) or na nocrystals has also been shown to
effectively improve their optical properties (Wei et al.,2007) .

3 Application s of near -infrared fluorescence immunoassay

3.1 Antibiotic test
In 2016, Chen et al. developed a multiple lateral flow immunoassay based on near -infrared
fluorescence by combining nea r-infrared labeling with broad -spectrum -specific monoclonal
antibody/receptor as a detection complex. The method can simultaneously detect four antibiotic
families in milk such as β-lactams, tetracyclines, quinolones and sulfonamides within 20
minutes( Chen et al.,2016)

3.2 Medical diagnostic marker molecular detection

Near-infrared fluorescence immunoassay

Compared with a few reports of near -infrare d fluorescence immunoassay for antibiotic test , this
method is more widely used in immunoassay of important marker molecules in diagnostics. So far,
near-infrared fluorescent probes have been successfully dev eloped for immunotiter plates,
fiber-optic immunosensors ( Daneshvar et al.,1999) capillary blotting (Zhao et al.,2004) , capillary
electrophoresis immunoassays (Liu et al.,2003) and immunochromatog raphic assays. A variety of
different immuno assay models such as strips ( Swanson et al.,2013) , are used to detect key proteins
for medical diagnosis.
In the early 1990s, NIR fluorescent probes were first applie d in immunoassays . Researchers
achieved quanti tative determination of human immunoglobulin by adding an excess of
NIR-labeled antibody and subsequent fluorescence detection in an antigen -coate d polyethylene
microtiter plate (Boyer et al., 1992). Daneshvar et al. ( Daneshvar et al.,1996) designed and
developed a fluorescent fiber -optic immunosenseor (FFOI) for near -infrared labeling of human
IgG. An a ntibody is immobilized on the sensing end of FFOI for the identification and capture of
trace specific antigens. The immune mode is sandwich type, which can be completed within
10~15 min with a detection limit of 10 ng/mL. In a follow -up study, the Dye1 used in the above
studies was replaced by a water -soluble NIR dye. The FFOI system was further confirmed to
efficiently quantify human IgG and effectively sen sitize one order of magnitude, while FFOI can
also be used in the detection of Legionella pneumophila (Daneshvar et al.,1999). The detection
sensitivity of the FFOI system is comparable to that of E LISA, and it has many advantages such as
short operative ti me, low detection cost and suitable for on -site detectio n. In addition, Silva et al.
(Silva 2004) developed an optical immunosensor based on the near -infrared dye Cy5 for the
determination of disease infection in sheep Brucella sp., which can achieve Bruce lla sp. antibody
in serum of sick sheep (0.005~0.11 mg/mL) q uantitative analysis. According to the difference in
electrophoretic behavior between antigen -antibody complexes and free antigens and antibodies,
Cy5 was also used for capillary electrophoresis i mmunoassay of IgA secreted in human saliva .
In recent years, novel NIFPs other than organic fluorescent dyes have also been introduced into
near-infrared fluorescent immunoassay syste ms. For example, Deng et al. ( Deng et al.,2006)
prepared a novel core/sh ell NIF nanoparticle by encapsulating the inexpensive near -infrared
fluorescent dye methylene blue in a hydrophobic silica gel shell. The immunoglobulin was used to
determine the alpha fetus in the whole blood sample. protein. This special structure exhibi ts higher
fluorescence intensity and better stability than conventional dye -coated silicon nanoparticles,
thereby preventing interference from dye leakage and exogenous quenching factors. In addition,
dual stabilizer -modified CdTe ( Liang et al.,2014) , CdTe /CdS core (thin) / shell (thick) (Wang et
al.,2012) and CdTe ( Liang et al.,2009) and CdSeTe/CdS/ZnS with mercaptop ropionic acid as
stabilizers (Li et al.,2013) quantum dot near -infrared electrochemiluminescence immunosensors
have also been developed for th e detection of fetal protein antigens, human IgG and
carcinoembryonic antigens respectively. The above system utilizes a near -infrared fluorescence
resonance energy transfer system to measure the distance effect caused by the immune reaction
between a near -infrared quantum dot -labeled protein and another probe (such as gold particles),
and the fluorescence is caused by the induced energy transfer. The change in intensity enables
high-sensitivity quantitative detection of target analytes. In addition to NIR -QDs, the emerging
NIR fluorescent material SWCNTs have also been used in IgG i mmunoa ssays by Iizumi et al.
(Iizumi et al.,2013). By detecting co -immunoprecipitation between IgG -bound SWCNTs and
immunomagn etic beads linked to protein G, the system can measure target analytes at

Near-infrared fluorescence immunoassay

concentrations as low as 600 pmol/L.
One of the early pathological markers of Alzheimer's disease (AD) is the deposition of
amyloid -beta (Aβ) plaque s in the brain. Optical imaging and particularly near -infra red
fluorescence (NIRF) imagi ng has become a safe, low -cost, real -time and widely available
technique that provides an attractive method for in vivo detection of A βplaques in many different
imaging techniques. In 2015, Tong et al. briefly outlined the latest developments in NIRFA β
probes and their applications in vitro and in vivo (Tong et al.,2015) .
Despite its high sensitivity, applications of NIFPs in immunoassays are still lacking in
simplicity, so its user friendliness needs further improvement. To overcome this short coming,
Swans on and D Andrea developed quantitative immunochromatographic test strip s( ICTSs )based
on NIFPs for single – and multiple -synchronous detection of interleukin -6 and C -reactive protein .
In 2013, Swanson developed a quantitative ICTSs based on a near -infrare d fluorescent probe. The
NIR dye was coupled to the selected antibody and integrated into the LFA. The test strip can
detect single and multiple simultaneous detection of interl eukin -6 and C -reactive protein
(Swanson et al.,2013) . The high signal -to-noise ratio of NIFPs makes the detection limit of the test
strip as low as pg/mL .That is e quivalent to ELISA. In summary, NIR -labeled ICTSs provide a
powerful tool for the evaluation of biomarker proteins in a real -time assay environment.

3.3 Application s in microorganism
Although current in vitro and in vivo bio -imaging is still the main application field of NIFPs , its
application in immunoassay has never stopped for many years since Boyer was the first researcher
in 1992 (Boyer et al., 1992). With continuous exploration of new NIFPs and continuous
development of immunoassay technology, the combination of the two has become increasingly
popular in many analytical fields. LFA is the most powerful immunoassay for immediate testing
due to its simple operation and portability. The application of NIFPs in LFA will undoubtedly
provide a valuable platform on -site, high -sensitivity bioanalysis in the near future.
In 2013, Cheng et al extracted the anti -pulmonary Legionella LP antigen to prepare
immunofluorescent LP ant ibody kit, and explored application value of near -infrared fluorescence
detection of Legionella pneumophila (LP) antigen (Cheng et al.,2013) . It was found that this
method is not related to other common bacteria. Cross -reactivity occurs with a minimum of 10
ng/ml, with good stability and repeatability. In recent years, some scholars have combined
near-infrared technology with immunomagnetic bead coupling method for quantitative detection.
Zhou et al (Zhou et al.,2015) labeled the monoclonal antibody targetin g larabinomannan (LAM)
with a near -infrared fluorescent dye, and the L AM method for detecting Mycobacterium
tuberculosis by targeting the multi -antibody of LAM coated on the surface of the nanomagnetic
beads. They used a double antibody sandwich method to magnetically separate the conjugate and
the free substance, and then used a portable near -infrared fluorescence detector to detect the
fluorescence intensity of the magnetic conjugate, thereby detecting the LAM content in the sample
to be tested, and foun d that the minimum detection limit of the metho d was 0.5 ng/mL. . In 2018,
Lin Chen et al. developed a new lateral flow assay (LFA) based on near -infrared (NIR) fluorescent
dyes to detect anti -dengue virus (DENV1) IgG antibodies. The results of NIR -LFA wer e compared
to those of Panbio Dengue IgG ELISA and the Dengue Duo IgM / IgG Kit. They identified 19
confirmed DENV1 positive samples by NIR -LFA with 95% sensitivity (Chen et al., 2018) .

Near-infrared fluorescence immunoassay

3.4 Our team's research on near-infrared fluorescence immunoassay
Foodborne pathogens are one of the most important threat factors for food poisoning incidents
worldwide. However, traditional microbial culture -based assays are time consuming and labor
intensive, failing to provide timely data to effectively reduce the inci dence of foodborne illness.
Therefore, Whether it is from the control of product quality by food companies, or the
government's effective supervision of food safety, so as to protect public health, we urgently need
a faster and independent method for detec ting food -borne pathogenic microorganisms. Although
current colloidal gold -based LFA are still the gold standard for rapid detection of pathogens , the
labeling technique is still limited by its low sensitivity and inability to accurately quantify defects.
Fluorescent probes are methods that use optical properties of fluorescent molecules to study some
of the physical, chemical, and physical properties of a particular environmental material at the
molecular level. It has high sensitivity and wide dynamic res ponse range, so it is widely used in
biological macromolecules. In the near -infrared region, biomolecules have weak self -fluorescence
and small background interference, and high sensitivity is obtained in this region. Therefore, the
research of NIFPs has b ecome a research hotspot in recent years, showing great potential in
biological analysis.
At present , our team has developed a n approach for detecting pathogen s such as Salmonella,
Vibrio parahaemolyticus, Vibrio cholerae and Listeria monocytogenes by near -infrared
immunoassay . In 2017, we used near infrared fluorescent marker of Vibrio parahaemolyticus
monoclonal antibody, Vibrio parahaemolytic us polyclonal antibody and Goat anti mouse IgG
polyclonal antibody was coated on nitrocellulose membrane as the de tection line and the control
line. We has developed the detection of Vibrio parahaemolyticus near infrared later flow assay
strips and supported the standard substance. The results showed that the near infrared
spectroscopy technique has good specificity a nd high sensitivity for Vibrio parahaemolyticus , the
lowest detection limit is 1.2*102 CFU/mL. No cross -reaction with Salmonella , Staphylococcus
aureus, Escherichia coli and Listeria monocytogenes . Compared with the traditional detection
method, the detect ion time of near infrared fluorescence method is the shortest, and the detection
limit is close to that of RT -PCR method. This near infrared immunochromatographic method is
ompleted in 45 minutes . It c ould be used for efficient detection of Vibrio parahae molyticus in
food and provide reliable technical support for food safety supervision .

4 Conclusions and Prospect s

At present, near -infrared spectroscopy technology has developed with d evelopment of different
fields such as computer science, chemometrics, photomaterial sc ience and measuring instruments ,
and has become a widely used analytical tool to solve some difficulties for quick detection
challenges. This technology requires qualitative and quantitative analysis of unknown samples by
establishing a ca libration model. Because the absorption spectrum is a superposition of the
absorption spectra of the contained compounds, and the map has a certain similarity, the map is
complicated and difficult to resolve. The further popularization of near -infrared spe ctroscopy in
the field of detection will be a great challenge. Therefore, near-infrared spectroscopy technology
needs to be combined with characteristics of actual detection to develop near-infrared
spectroscopy special information of each system and the p rocessing technology of other
disciplines, so that it can be better applied. With the development of NIR analysis technology and

Near-infrared fluorescence immunoassay

other fields of technology and the continuous expansion of its application, it will play an important
role in analytical tools modernization .
Since labeled molecules play a decisive role in the development of immunochromatography
technology platforms, it is particularly important to develop ideal probes with high quantum yield,
good stability, small background interference and ea sy labeling. The existing probe development
mostly relies on simplicity to obtain high sensitivity and accuracy, which to some extent
undermines the superiority of ICTSs in the field and in real -time detection. Therefore, more
innovative research should be devoted to the development of highly accurate probes . At present
near-infrared fluorescent probe has excellent potential, but it is mainly used for biological imaging
research, and the application on the ICTSs is almost blank. Previous studies have shown that
near-infrared fluorescent probe labels are 100 times more effective than traditional colloidal gold
test strips in detecting HIV.
In recent years, our team has developed a method for detecting pathogen s include d Salmonella,
Vibrio parahaemolyticus, V ibrio cholerae and Listeria monocytogenes by near -infrared
immunoassay . Our team is working on food -borne viruses such as rotavirus, norovirus an d
hepatitis A virus , and we could develop near-infrared ICTSs using NIFPs -labeled antibodies to
detect these fo odborne viruses. Our team intends to establish a new platform which has a highly
sensitive near infrared probe combined with portable and simple ICTSs for rapid detection of food
borne viruses. This will provide technical support for a rapid detection on t he port .

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