Design and synthesis of a novel fluorescent protein probe for easy and rapid electrophoretic gel staining by using a commonly available UV‐based fluorescent imaging system

A new fluorescent molecular probe, methyl 3‐(3,5‐bis((bis(pyridin‐2‐ylmethyl)amino)‐methyl)‐4‐hydroxyphenyl)‐2‐(5‐(dimethylamino)naphthalene‐1‐sulfonamido) propanoate, dizinc(II) chloride salt (Dansyl‐ 1 ‐Zn(II)), which possesses Zn(II) complexes and a dansyl group, was designed and synthesized to enable the detection of proteins in solution and in high‐throughput electrophoresis by using a UV‐based detection system. Dansyl‐ 1 ‐Zn(II) exhibited weak fluorescence in the absence of proteins and strong green fluorescence at approximately 510 nm in the presence of BSA upon irradiation with light at a wavelength of 345 nm. Compared with conventional protocols for in‐gel SDS‐PAGE protein staining (e.g. silver staining, SYPRO Ruby, and Oriole), the operating times of which range from 90 min to overnight, Dansyl‐ 1 ‐Zn(II) allowed 1‐step protein staining (SDS‐PAGE →Staining →Detection) and shortened the operating time (35 min) with high sensitivity (LOD: 1 ng or less) under 312‐nm or 365‐nm light excitation with orange or red emission filters, respectively. Moreover, Dansyl‐ 1 ‐Zn(II) was successfully applied to protein identification by MS via in‐gel tryptic digestion, Western blotting, and Native‐PAGE. Accordingly, Dansyl‐ 1 ‐Zn(II) may facilitate highly sensitive and high‐throughput protein detection, and it may be widely applicable as a convenient tool in various scientific and medical fields.     

Improved conditions for fluorescent staining of proteins with 4,4′‐dianilino‐1,1′‐binaphthyl‐5,5′‐disulfonic acid in SDS‐PAGE

A simple and sensitive fluorescent staining method for the detection of proteins in SDS‐PAGE, namely IB (improved 4,4′‐dianilino‐1,1′‐binaphthyl‐5,5′‐disulfonic acid) stain, is described. Non‐covalent hydrophobic probe 4,4′‐dianilino‐1,1′‐binaphthyl‐5,5′‐disulfonic acid was applied as a fluorescent dye, which can bind to hydrophobic sites in proteins non‐specifically. As low as 1 ng of protein band can be detected briefly by 30 min washing followed by 15 min staining without the aiding of stop or destaining step. The sensitivity of the new presented protocol is similar to that of SYPRO Ruby, which has been widely accepted in proteomic research. Comparative analysis of the MS compatibility of IB stain and SYPRO Ruby stain allowed us to address that IB stain is compatible with the downstream of protein identification by PMF.     

Postelectrophoretic staining of proteins separated by two-dimensional gel electrophoresis using SYPRO dyes

While the classical silver stain has been the method of choice for high sensitivity protein visualization on two-dimensional gel electrophoresis (2-D PAGE), post-electrophoretic fluorescent staining with the SYPRO group of dyes has emerged to challenge silver staining for proteome analysis. The latter offers improved sensitivity, higher dynamic range and easy handling. However, most of the published data were derived from analysis of 1-D gel separations. In this work, we have focused on three commercially available fluorescent dyes, SYPRO Ruby, SYPRO Orange and SYPRO Red (Molecular Probes, Eugene, OR, USA) and studied their sensitivity and dynamic range on 2-D PAGE. The use of a multiwavelength fluorescent scanner to image 2-D protein profiles visualized with fluorescent staining is discussed, and a detailed comparison with analysis by silver staining is also provided. These results demonstrate the advantages of using SYPRO dyes, which are in agreement with the literature based on 1-D gel electrophoresis, and give a more realistic understanding of the performance of these fluorescent dyes with 2-D PAGE.     

High-sensitivity staining of proteins for one- and two-dimensional gel electrophoresis using post migration covalent staining with a ruthenium fluorophore

This paper describes the use of a ruthenium complex ((bis(2,2'-bipyridine)-4'-methyl-4-carboxybipyridine-ruthenium-N-succidimyl ester-bis(hexafluorophosphate), abbreviated below as ASCQ_Ru) commercially available and chemically pure. This new ruthenium complex ASCQ_Ru brings an activated ester, allowing the selective acylation of amino acid side chain amines for the post migration staining of proteins separated in 1-DE and 2-DE. The protocol used is a simple three-step protocol fixing the proteins in the gel, staining and then washing, as no lengthy destaining step is required. First the critical staining step was optimized. Although in solution the best described pH for acylating proteins with this reagent is phosphate buffer at pH 7.0, we found that best medium for in-gel staining is unbuffered ACN/water solution (20/80 v/v). The two other steps are less critical and classical conditions are satisfactory: fixing with 7% acetic acid/10% ethanol solution and washing four times for 10 min with water. Sensitivity tests were performed using 1-DE on protein molecular weight markers. We obtained a higher sensitivity than SYPRO Ruby with a detection limit of 80 pg of protein per well. However, contrary to SYPRO Ruby, ASCQ_Ru exhibits a logarithmic dependency on the amount of protein. The dynamic range is similar to SYPRO Ruby and is estimated between three and four orders of magnitude. Finally, the efficiency of the post migration ASCQ_Ru staining for 2-D gel separation is demonstrated on the whole protein extract from human colon carcinoma cells lines HCT 116. ASCQ_Ru gave the highest number of spot detected compared to other common stains Colloidal CBB, SYPRO Ruby and Deep Purple.     

2-D native-PAGE/SDS-PAGE visualization of an oligomer's subunits: application to the analysis of IgG

A 2-D native-PAGE/SDS-PAGE method for detecting the subunit components of protein oligomers at low picomole sensitivity is presented. IgG was electrophoresed in a native acidic polyacrylamide gel in amounts ranging from 51 pmol to 60 fmol. Silver-staining (native fast silver stain, ammoniacal silver stain, permanganate silver stain), Coomassie-staining (R-250, G-250), metal ion-reverse-staining (zinc, copper), and fluorescent chromophore-staining (SYPRO Ruby) methods were used to visualize the IgG oligomers. The protein zones were then excised, separated by SDS-PAGE, and subunits visualized with a permanganate silver stain. The Coomassie R-250/permanganate silver-staining combination detected IgG subunits using 2 pmol of sample. Coomassie G-250 and native fast silver staining in the first-dimensional gel produced detectable subunits in the second-dimensional separation at 3 and 13 pmol, respectively. Staining with silver (ammoniacal, permanganate), copper, zinc, or SYPRO Ruby in the first-dimensional gel did not produce discernible subunits in the second-dimensional gels due to protein streaking or protein immobilization in the native gel. When using a 2-D native-PAGE/SDS-PAGE system, Coomassie staining of the first-dimensional native gel combined with permanganate silver staining of the second-dimensional denaturing gel provides the most sensitive method (2-3 pmol) for visualizing constituent subunits from their oligomeric assemblies.     

Comparison of fluorescent stains: relative photostability and differential staining of proteins in two-dimensional gels

The fluorescence of proteins stained with Deep Purple and SYPRO Ruby was measured over a time course of UV transillumination to determine the relative photostability of each stain. Mean spot fluorescence (n = 200 matched spots) in gels stained with Deep Purple decreased 27% following 2 min of UV transillumination, compared to SYPRO Ruby, which decreased 17%. After 19 min, an 83% decrease in Deep Purple fluorescence was observed, compared to 44% for SYPRO Ruby. By interpolation, the half-life of Deep Purple fluorescence was estimated to be approximately 6 min. The half-life of SYPRO Ruby fluorescence was not reached during the 19 min time course. Further, differential staining of proteins was observed in gels stained with Deep Purple and SYPRO Ruby as compared to colloidal Coomassie Brilliant Blue and silver staining.     

Rapid and easy protein staining for SDS-PAGE using an intramolecular charge transfer-based fluorescent reagent

High-performance staining for 1-D and 2-D SDS-PAGE was carried out using a novel protein-binding fluorophore (Dye 1), which noncovalently interacts with proteins and provides a fluorescence emission response to proteins by intramolecular charge transfer. In order to achieve the high-throughput analysis of proteins for SDS-PAGE, the general protocols for in-gel protein staining (SDS-PAGE, fixation, staining, washing, and detection) were simplified to produce an easy and rapid protocol (SDS-PAGE together with staining, washing, and detection). This method was performed by preparation of an electrophoresis buffer containing Dye 1 under optimum conditions, and by the binding of Dye 1 to proteins in the gel during the SDS-PAGE. As a result, this study required only 15 min for protein staining as a minimum time. On the other hand, it takes several hours for the general protein staining method, such as SYPRO Ruby staining (18 h) and CBB staining (105 min). Moreover, the protein-to-protein variation was low, and the detection limit was 7.0 ng/band of BSA (S/N = 3.0) in this method, which was as sensitive as the short-protocol silver staining methods. On the basis of these results, this rapid and easy protocol for SDS-PAGE using Dye 1 may be widely applicable and convenient for users in the various scientific and medical fields.     

Simultaneous, two-color fluorescence detection of total protein profiles and beta-glucuronidase activity in polyacrylamide gel

A dichromatic method for measuring the specific activity of beta-glucuronidase from complex cell homogenates or partially purified protein fractions is presented. Dual fluorescence is achieved by using the green emitting fluorogenic substrate ELF 97 beta-D-glucuronide to detect beta-glucuronidase activity, followed by the red emitting SYPRO Ruby protein gel stain or SYPRO Ruby IEF gel stain to detect the remaining proteins in the electrophoretic profile. Both ELF 97 alcohol, the highly fluorescent hydrolytic product generated from the enzyme substrate, and the SYPRO Ruby total protein stains are maximally excited by ultraviolet illumination. ELF 97 alcohol emits maximally at 525 nm while the SYPRO Ruby dyes emit maximally at 610 nm. Since ELF 97 beta-glucuronide is a precipitating substrate, it allows precise localization of beta-glucuronidase activity with minimal band diffusion. The staining method is simple and direct, without the requirement for ancillary coupling reactions. Dichromatic protein detection is demonstrated after sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis, carrier ampholyte-mediated isoelectric focusing or two-dimensional gel electrophoresis.     

Dual selective nitration in Arabidopsis: Almost exclusive nitration of PsbO and PsbP,and highly susceptible nitration of four non‐PSII proteins,including peroxiredoxin II E

Protein tyrosine nitration is a selective process, as revealed in studies of animals. However, evidence for selective protein nitration in plants is scarce. In this study, Arabidopsis plants were exposed to air with or without nitrogen dioxide at 40 ppm for 8 h in light. Proteins extracted from whole leaves or isolated chloroplasts were subjected to 2D PAGE followed by SYPRO Ruby staining and immunoblotting using an anti‐3‐nitrotyrosine antibody. We determined the relative intensity of a spot on an immunoblot (designated RISI), and relative intensity of the corresponding spot on SYPRO Ruby gel (designated RISS). Proteins that exhibited a high RISI value and/or a high RISI/RISS ratio were considered selectively nitrated. In whole leaf proteins from exposed plants, all immunopositive spots were identified as PsbO1, PsbO2 or PsbP1 by PMF. Thus, nitration was exclusive to PsbO and PsbP, extrinsic proteins of photosystem II (PSII). Their RISI/RISS ratio was ≤1.5. Non‐exposed plants showed very faint nitration. In purified chloroplast proteins, PsbO and PsbP accounted for >80% of the total RISI values, while four non‐PSII proteins, including peroxiredoxin II E, exhibited high RISI/RISS ratios (2.5～6.6). Tyr⁹ of PsbO1 was identified as a nitration site. Thus, nitration is selective for two PSII and four non‐PSII proteins in Arabidopsis.     

A statistical comparison of silver and SYPRO Ruby staining for proteomic analysis

Silver staining has been the method most commonly employed for high sensitivity staining of proteins following two-dimensional gel electrophoresis. Whilst this method offers detection in the nanogram range it does have major drawbacks including a lack of linearity, nonstoichiometric staining of proteins, a lack of compatibility with the microchemical preparation of proteins for identification by mass spectrometric techniques, and a highly subjective assessment of the staining endpoint. SYPRO Ruby is a relatively new, ruthenium complex-based stain which is reported to offer advantages over silver, particularly in overcoming the limitations cited above. We describe a series of experiments where several protein staining procedures commonly employed are compared. To enable optimization of the in situ digestion procedure, a statistical approach has been undertaken. The effects of a variety of staining, digestion, and analysis protocols on the downstream processing of a test radiolabeled protein were studied. The data confirms that as well as offering sensitivity similar to silver, SYPRO Ruby staining is reproducible, linear, and offers a higher level of compatibility with the identification of proteins by mass spectrometry.     

Practical aspects of fluorescent staining for proteomic applications

SYPRO Orange and SYPRO Ruby staining methods, modified for use with large-format two dimensional (2-D) gels, are compared to the manufacturer's recommended protocols to determine sensitivity and reproducibility of the new methods. This study examines the critical aspects of fixation, washing, and staining to develop an optimized fluorescent staining method. It was determined that careful control of sodium dodecyl sulfate (SDS) levels and pH in the gel was critical for successful staining with SYPRO Orange. Overnight fixation in 40% ethanol/2% acetic acid/0.0005% SDS preserved protein content, eliminated ampholyte-generated staining artifacts, and had no detrimental effects on staining. Three one-hour washes in 2% acetic acid/0.0005% SDS, followed by staining with SYPRO Orange diluted 1:5,000 with washing solution for 3 or more hours, produced high sensitivity, low background images using a STORM 860 laser scanner. Gels viewed two years after staining showed no significant changes with respect to the initial protein patterns, and allowed successful mass spectrometric postgel characterization of protein spots. Protocol changes applied to SYPRO Ruby staining improved the contrast of STORM 860-generated images, but had little impact on staining sensitivity. A comparison of the cost benefits of staining with SYPRO Orange vs. SYPRO Ruby is also discussed.     

Fluorescent staining of protein in SDS polyacrylamide gels by salicylaldehyde azine

As a noncovalent fluorescence probe, in this study, salicylaldehyde azine (SA) was introduced as a sensitive fluorescence‐based dye for detecting proteins both in 1D and 2D polyacrylamide electrophoresis gels. Down to 0.2 ng of single protein band could be detected within 1 h, which is similar to that of glutaraldehyde‐silver stain, but approximately four times higher than that of SYPRO Ruby fluorescent stain. Furthermore, comparative analysis of the MS compatibility of SA stain with SYPRO Ruby stain indicated that SA stain is compatible with the downstream of protein identification by LC‐MS/MS. Additionally, the probable mechanism of the SA stain was investigated by molecular docking. The results demonstrated that the interaction between SA and protein was mainly contributed by hydrogen bonding and hydrophobic forces.     

Optimized conditions for diluting and reusing a fluorescent protein gel stain

This study elucidates the optimum conditions at the minimum cost for using SYPRO Ruby protein gel stain. It deals with the effects of gel fixation and staining times, as well as dilution and reuse of SYPRO Ruby protein gel stain in one-dimensional (1-D) gels. Signal strength and dynamic range were highest in gels that were fixed thoroughly before staining, followed by overnight staining. Using the optimized protocol, dilution or reuse of the stain reduces the dynamic range and signal intensity. Sensitivity remains high if the stain is reused up to two times, but signal intensity is reduced up to 2.5-fold in twice used stain. Sensitivity also remains high if the stain is diluted 1:2 in water, but signal intensity is reduced up to 6-fold. Of the two options, reuse or dilution, reuse better retains signal intensity and dynamic range.     

Ultrasensitive fluorescence protein detection in isoelectric focusing gels using a ruthenium metal chelate stain

SYPRO Ruby IEF Protein Gel Stain is an ultrasensitive, luminescent stain optimized for the analysis of protein in isoelectric focusing gels. Proteins are stained in a ruthenium-containing metal complex overnight and then rinsed in distilled water for 2 h. Stained proteins can be excited by ultraviolet light of about 302 nm (UV-B transilluminator) or with visible light of about 470 nm. Fluorescence emission of the dye is maximal at approximately 610 nm. The sensitivity of the SYPRO Ruby IEF protein gel stain is superior to colloidal Coomassie blue stain and the highest sensitivity silver staining procedures available. The SYPRO Ruby IEF protein gel stain is suitable for staining proteins in nondenaturing or denaturing carrier ampholyte isoelectric focusing and immobilized pH gradient gel electrophoresis. The stain is compatible with N,N'-methylenebisacrylamide or piperazine diacylamide cross-linked polyacrylamide gels as well as with agarose gels and high tensile strength Duracryl gels. The stain does not contain extraneous chemicals (formaldehyde, glutaraldehyde, Tween-20) that frequently interfere with peptide identification in mass spectrometry. Successful identification of stained proteins by peptide mass profiling is demonstrated.     

Quantitative evaluation of proteins in one- and two-dimensional polyacrylamide gels using a fluorescent stain

The characteristics of protein detection and quantitation with SYPRO Ruby protein gel stain in one- and two-dimensional polyacrylamide gels were evaluated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of three different purified recombinant proteins showed that the limits of detection were comparable to the limits of detection with ammoniacal silver staining and were protein-specific, ranging from 0.5 to 5 ng. The linearity of the relationship between protein level and SYPRO Ruby staining intensity also depended on the individual protein, with observed linear dynamic ranges of 200-, 500-, and, 1000-fold for proteins analyzed by SDS-PAGE. SYPRO Ruby protein gel stain was also evaluated in two-dimensional electrophoretic (2-DE) analysis of Escherichia coli proteins. The experiment involved analysis of replicates of the same sample as well as dilution of the sample from 0.5 to 50 nug total protein across gels. In addition to validating the 2-DE system itself, the experiment was used to evaluate three different image analysis programs: Z3 (Compugen), Progenesis (Nonlinear Dynamics), and PDQuest (Bio-Rad). In each program, we analyzed the 2-DE images with respect to sensitivity and reproducibility of overall protein spot detection, as well as linearity of response for 20 representative proteins of different molecular weights and pI. Across all three programs, coefficients of variation (CV) in total number of spots detected among replicate gels ranged from 4 to 11%. For the 20 representative proteins, spot quantitation was also comparable with CVs for gel-to-gel reproducibility ranging from 3 to 33%. Using Progenesis and PDQuest, a 1000-fold linear dynamic range of SYPRO Ruby was demonstrated with a single known protein. These two programs were more suitable than Z3 for examining individual protein spot quantity across a series of gels and gave comparable results.     

A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry

The growing availability of genomic sequence information, together with improvements in analytical methodology, have enabled high throughput, high sensitivity protein identification. Silver staining remains the most sensitive method for visualization of proteins separated by two-dimensional gel electrophoresis (2-D PAGE). Several silver staining protocols have been developed which offer improved compatibility with subsequent mass spectrometric analysis. We describe a modified silver staining method that is available as a commercial kit (Silver Stain PlusOne; Amersham Pharmacia Biotech, Amersham, UK). The 2-D patterns abtained with this modified protocol are comparable to those from other silver staining methods. Omitting the sensitizing reagent allows higher loading without saturation, which facilitates protein identification and quantitation. We show that tryptic digests of proteins visualized by the modified stain afford excellent mass spectra by both matrix-assisted laser desorption/ionization and tandem electrospray ionization. We conclude that the modified silver staining protocol is highly compatible with subsequent mass spectrometric analysis.     

Synthesis and characterization of alt‐bipyridinylene‐phenylene copolymers containing ruthenium(II) complexes

Poly{bis(4,4′‐tert‐butyl‐2,2′‐bipyridine)–(2,2′‐bipyridine‐5,5′‐diyl‐[1,4‐phenylene])–ruthenium(II)bishexafluorophosphate} ( 3a ), poly{bis(4,4′‐tert‐butyl‐2,2′‐bipyridine)–(2,2′‐bipyridine‐4,4′‐diyl‐[1,4‐phenylene])–ruthenium(II)bishexafluorophosphate} ( 3b ), and poly{bis(2,2′‐bipyridine)–(2,2′‐bipyridine‐5,5′‐diyl‐[1,4‐phenylene])–ruthenium(II)bishexafluorophosphate} ( 3c ) were synthesized by the Suzuki coupling reaction. The alternating structure of the copolymers was confirmed by ¹H and ¹³C NMR and elemental analysis. The polymers showed, by ultraviolet–visible, the π–π* absorption of the polymer backbone (320–380 nm) and at a lower energy attributed to the d–π* metal‐to‐ligand charge‐transfer absorption (450 nm for linear 3a and 480 nm for angular 3b ). The polymers were characterized by a monomodal molecular weight distribution. The degree of polymerization was approximately 8 for polymer 3b and 28 for polymer 3d . © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 2911–2919, 2004     

The development of simple and sensitive small-molecule fluorescent probes for the detection of serum proteins after native polyacrylamide gel electrophoresis

In this paper, a simple and sensitive small-molecule fluorescent probe, 2,5-dihydroxy-4'-dimethylaminochalcone (DHDMAC), was designed and synthesized for the detection of human serum proteins via hydrophobic interactions after polyacrylamide gel electrophoresis (PAGE). This probe produced lower fluorescence emission in the absence of proteins, and the emission intensity was significantly increased after the interaction with serum proteins. To demonstrate the imaging performance of this probe as a fluorescent dye, a series of experiments was conducted that included sensitivity comparison and 2D-PAGE. The results indicated that the sensitivity of DHDMAC staining is comparable to that of the most widely used fluorescent dye, SYPRO Ruby, and more protein spots (including thyroxine-binding globulin, angiotensinogen, afamin, zinc-α-2-glycoprotein and α-1-antichymotrypsin) were detected after 2D-PAGE. Therefore, DHDMAC is a good protein reporter due to its fast staining procedure, low detection limits and high resolution.     

Background-free, high sensitivity staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gels using a luminescent ruthenium complex

SYPRO Ruby dye is a permanent stain comprised of ruthenium as part of an organic complex that interacts noncovalently with proteins. SYPRO Ruby Protein Gel Stain provides a sensitive, gentle, fluorescence-based method for detecting proteins in one-dimensional and two-dimensional sodium dodecyl sulfate-polyacrylamide gels. Proteins are fixed, stained from 3h to overnight and then rinsed in deionized water or dilute methanol/acetic acid solution for 30 min. The stain can be visualized using a wide range of excitation sources commonly used in image analysis systems including a 302 nm UV-B transilluminator, 473 nm second harmonic generation (SHG) laser, 488 nm argon-ion laser, 532 nm yttrium-aluminum-garnet (YAG) laser, xenon arc lamp, blue fluorescent light bulb or blue light-emitting diode (LED). The sensitivity of SYPRO Ruby Protein Gel Stain is superior to colloidal Coomassie Brilliant Blue (CBB) stain or monobromobimane labeling and comparable with the highest sensitivity silver or zinc-imidazole staining procedures available. The linear dynamic range of SYPRO Ruby Protein Gel stain extends over three orders of magnitude, which is vastly superior to silver, zinc-imidazole, monobromobimane and CBB stain. The fluorescent stain does not contain superfluous chemicals (formaldehyde, glutaraldehyde, Tween-20) that frequently interfere with peptide identification in mass spectrometry. While peptide mass profiles are severely altered in protein samples prelabeled with monobromobimane, successful identification of proteins by peptide mass profiling using matrix-assisted laser desorption/ionization mass spectrometry was easily performed after protein detection with SYPRO Ruby Protein Gel stain.     

A New Chiral Binaphthalene‐Based Fluorescence Polymer Sensor for the Highly Enantioselective Recognition of Phenylalaninol

A new (S)‐binaphthalene‐based polymer ( P ‐ 1 ) was synthesized by the polymerization of 5,5′‐((2,5‐dibutoxy‐1,4‐phenylene)bis(ethyne‐2,1‐diyl))bis(2‐hydroxy‐3‐(piperidin‐1‐ylmethyl) benzaldehyde ( M ‐ 1 ) with (S)‐2,2′‐dimethoxy‐(1,1′‐binaphthalene)‐3,3′‐diamine ( M ‐ 2 ) through the formation of a Schiff base; the corresponding chiral polymer ( P ‐ 2 ) could be obtained by the reduction of polymer P ‐ 1 with NaBH₄. Chiral polymer P ‐ 1 exhibited a remarkable “turn‐on” fluorescence‐enhancement response towards (D )‐phenylalaninol and excellent enantioselective recognition behavior with enantiomeric fluorescence difference ratios (ef) as high as 8.99. More importantly, chiral polymer P ‐ 1 displays a bright blue fluorescence color change upon the addition of (D )‐phenylalaninol under a commercially available UV lamp, which can be clearly observed by the naked eye. On the contrary, chiral polymer P ‐ 2 showed weaker enantioselective fluorescence ability towards the enantiomers of phenylalaninol.